U.S. patent number 6,309,669 [Application Number 08/789,734] was granted by the patent office on 2001-10-30 for therapeutic treatment and prevention of infections with a bioactive materials encapsulated within a biodegradable-biocompatible polymeric matrix.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the Army. Invention is credited to Edgar C. Boedeker, William Brown, Frederick Cassels, Phil Friden, Elliot Jacob, Daniel L. Jarboe, Ramasubbu Jeyanthi, Charles E. McQueen, Robert H. Reid, F. Donald Roberts, Jean A. Setterstrom, Curt Thies, Thomas R. Tice, John E. Van Hamont.
United States Patent |
6,309,669 |
Setterstrom , et
al. |
October 30, 2001 |
Therapeutic treatment and prevention of infections with a bioactive
materials encapsulated within a biodegradable-biocompatible
polymeric matrix
Abstract
Novel burst-free, sustained release biocompatible and
biodegrable microcapsules which can be programmed to release their
active core for variable durations ranging from 1-100 days in an
aqueous physiological environment. The microcapsules are comprised
of a core of polypeptide or other biologically active agent
encapsulated in a matrix of poly(lactide/glycolide) copolymer,
which may contain a pharmaceutically-acceptable adjuvant, as a
blend of upcapped free carboxyl end group and end-capped forms
ranging in ratios from 100/0 to 1/99.
Inventors: |
Setterstrom; Jean A.
(Alpharetta, GA), Van Hamont; John E. (Fort Meade, MD),
Reid; Robert H. (McComas, CT), Jacob; Elliot (Silver
Spring, MD), Jeyanthi; Ramasubbu (Columbia, MD),
Boedeker; Edgar C. (Chevy Chase, MD), McQueen; Charles
E. (Olney, MD), Jarboe; Daniel L. (Silver Spring,
MD), Cassels; Frederick (Ellicott City, MD), Brown;
William (Denver, CO), Thies; Curt (Ballwin, MO),
Tice; Thomas R. (Birmington, AL), Roberts; F. Donald
(Dover, MA), Friden; Phil (Beford, MA) |
Assignee: |
The United States of America as
represented by the Secretary of the Army (Washington,
DC)
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Family
ID: |
25148532 |
Appl.
No.: |
08/789,734 |
Filed: |
January 27, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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590973 |
Jan 24, 1996 |
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446149 |
May 22, 1995 |
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590308 |
Mar 6, 1984 |
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789734 |
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446148 |
May 22, 1995 |
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867301 |
Apr 10, 1992 |
5417986 |
May 23, 1995 |
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590308 |
Mar 16, 1984 |
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Current U.S.
Class: |
424/486; 424/422;
424/484; 424/425; 424/423; 424/424 |
Current CPC
Class: |
A61K
9/5031 (20130101); A61K 39/0258 (20130101); A61K
45/06 (20130101); A61K 9/5026 (20130101); A61K
38/09 (20130101); A61K 9/1647 (20130101); A61K
39/00 (20130101); A61K 38/1729 (20130101); A61K
39/12 (20130101); A61K 39/292 (20130101); A61K
38/09 (20130101); A61K 2300/00 (20130101); A61K
38/1729 (20130101); A61K 2300/00 (20130101); A61K
39/0258 (20130101); A61K 2300/00 (20130101); A61K
39/292 (20130101); A61K 2300/00 (20130101); A61K
9/1635 (20130101); Y02A 50/474 (20180101); A61K
2039/55505 (20130101); A61K 9/1611 (20130101); Y02A
50/48 (20180101); Y02A 50/464 (20180101); C12N
2730/10134 (20130101); Y02A 50/30 (20180101); A61K
9/1641 (20130101); A61K 9/1617 (20130101); A61K
2039/545 (20130101); A61K 2039/55555 (20130101) |
Current International
Class: |
A61K
9/16 (20060101); A61K 9/50 (20060101); A61K
38/17 (20060101); A61K 009/52 (); A61K
047/30 () |
Field of
Search: |
;424/78.17,78.08,422,423,424,425,484,486 ;514/2,772.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Wang et al. Influence of Formulation Methods on the in vitro
Controlled Release of Protein from Poly(ester) Microspheres. J. of
Controlled Release. Sep. 1991, vol. 17, pp. 23-31.* .
Yan et al. Characterization and Morphological Analysis of
Protein-Loaded Poly(Lactide-co-Glycolide) Microparticles Prepared
by WOW Emulsion Technique. J. of Con. Rel. 1994, 32(3). pp.
231-241.* .
Yeh et al. A Novel Emulsification-Solvent extraction Technique for
Production of Protein Loaded Biodegradable Microparticles for
vaccine and Drug Delivery. 1995, 33(3), pp. 437-445.* .
Jeyanthi et al. Novel, Burst-Free, Programmable Biodegradable
Microspheres for Controlled Release of Polypeptides. In:
Proceedings International Symposium on Controlled Release of
Bioactive.* .
Materials 1996. Pp. 351-352..
|
Primary Examiner: Harrison; Robert H.
Attorney, Agent or Firm: Nash; Caroline Arwine;
Elizabeth
Government Interests
I. GOVERNMENT INTEREST
The invention described herein may be manufactured, used and
licensed by or for the Government for Govermental purposes without
the payment to use of any royalties thereon.
Parent Case Text
II. CROSS REFERENCE
This application is a continuation-in-part of U.S. patent
application Ser. No. 08/590,973 filed Jan. 24, 1996 now abandoned
which in turn is a continuation-in-part of U.S. patent application
Ser. No. 08/446,149 filed May 22, 1995 now abandoned which in turn
is a continuation of U.S. patent application Ser. No. 06/590,308
dated Mar. 16, 1984 now abandoned.
Additionally, this application is a continuation-in-part of U.S.
patent application Ser. No. 08/446,148 filed May 22, 1995, which in
turn is a continuation-in-part of U.S. patent application Ser. No.
07/867,301 filed Apr. 10, 1992 now U.S. Pat. No. 5,417,986 issued
May 23, 1995, which in turn is a continuation-in-part of U.S.
patent application Ser. No. 06/590,308 filed Mar. 16, 1984 now
abandoned.
Claims
What we claim is:
1. A controlled release microcapsule phamaceutical formulation for
burst-free, sustained, programmable release of a biologically
active agent over a duration from 1-100 days, comprising an active
agent encapsulated within a biodegradable poly(lactide/glycolide)
having a lactide/glycolide ratio of 90/10 to 40/60 and the
uncapped/end capped form of said poly(lactide/glycolide) in the
ratio of 100/0 to 1/99, wherein the poly(lactide/glycolide) may
contain a pharmaceutically-acceptable adjuvant.
2. The composition of claim 1 wherein the copolymer (lactide to
glycolide L/G) ratio for uncapped and end-capped polymer is 48/52
to 52/48.
3. The composition of claim 1 wherein the molecular weight of the
poly(lactide/glycolide) is between 2,000-60,000 daltons.
4. The composition of claim 1 wherein the active material is a
biologically active agent.
5. The composition of claim 4 wherein the agent is selected from
the group consisting of antibacterial agents; peptides;
polypeptides; antibacterial peptides; antimycobacterial agents;
antimycotic agents; antiviral agents; antiparastic agents;
antifungal; hormonal peptides; hormonal peptides; cardiovascular
agents; narcotic antagonists; analgesics; anesthetics; insulins;
steroids; HIV therapeutic drugs; protease inhibitors; AZT;
estrogens; progestins; gastrointestinal therapeutic agents;
nonsteroidal anti-inflammatory agents; parasympathoimetic agents;
psychotherapeutic agents; tranquilizers; decongestants;
sedative-hypnotics; non-estrogenic and non-progestional steroids;
sympathomimetic agents; vaccines; vitamins; nutrients;
anti-migraine drugs; electrolyte replacements; ergot alkaloids;
anti-inflammary agents; prostaglandins; cytotoxic drugs; antigens;
antibodies; enzymes; growth factors; immunomodulators; pheromones;
prodrugs; psychotropic drugs; nicotine; antiblood clotting drugs;
appetite suppressants/stimulants and combinations thereof;
contraceptive agents; estrogens; diethyl silbestrol;
17-beta-estradiol; estrone; ethinyl estradiol; mestranol;
progestins; norethindrone; norgestryl; ethynodiol diacetate;
lynestrenol; medroxyprogesterone acetate; dimethisterone; megestrol
acetate; chlormadinone acetate; norgestimate; norethisterone;
ethisterone; melentate; melengestrol; norethynodrel; spermicidal
compounds; nonyphenoxypolyoxyethylene glycol; benzethonium
chloride; chlorindanol; gastrointestinal therapeutic agents;
aluminum hydroxide; calcium carbonate; magnesium carbonate; sodium
carbonate and the like; non-steroidal antifertility agents;
parasympathomimetic agents; psychotherapeutic agents; major
tranquilizers; chloropromaquine HCL; clozapine; mesoridazine;
metiapine; reserpine; thioridazine; minor tranquilizers;
chlordiazepoxide; diazepam; meprobamate; temazepam and the like;
rhinological decongestants; sedative-hypnotics; codeine;
phenobarbital; sodium pentobarbital; sodium secobarbital; other
steroids; testosterone; testosterone propionate; sulfonmides;
sympathomimetic agents; vaccines; vitamins and nutrient; the
essential amino acids; essential fats; anti-HIV agents; including
AZT; antimalarials; 4-aminoquinolines; 8 aminoquinolines;
pyrimethamine; anti-migraine agents; mazindol; phentermine;
anti-Parkinson agents; L-dopa; antispasmodics; atropine;
methscopolamine bromide; antispasmodics and anticholingeric agents;
bile therapy; digestants; enzymes and the like; antitussives;
dextromethorphan and noscapine; bronchodilators; anti-hypertensive
compounds; Rauwolfia alkaloids; coronary vasodilators;
nitroglycerin; organic nitrites; pentaerythriotetranitrate;
electrolyte replacements; potassium chloride; ergotalkaloids;
ergotamine with and without caffein; hydrogenated ergot alkaloids;
dihydroergocristine methanesulfate; dihydroergocornine
methanesulfonate; dihydroergokroyptine methaneusulfate and
combinations thereof; alkaloids; atropine sulfate; Belladonna;
hyoscine hydrobromide; analgesics; narcotics; codeine;
dihydrocodienone; meperidine; morphine; non-narcotics; salicylates;
aspirin; acetaminophen; d-propoxyphene; antibiotics;
cephalosporins; ceflacor; cefuroxime; chloranphenical; gentamicin;
Kanamycin A.; Kanamycin B; penicillins; ampicillin; amoxicillin;
streptomycin A; antimycin A; chloropamtheniol; metromidazole;
oxytetracycline penicillin G; tetracyclines; minocycline;
fluoro-quinolones; ciprofloxacin; ofoxacin; macrolides;
clarithromycin; frythromycin; aminioglycosides; gentamicin;
amikacin; tobramycin; kanamycin; beta-lactams; ampacillin;
polymyxin-B; amphotercin-B; aztrofonam; chloramphenicol; fusidans;
lincosamides; metronidazole; nitro-furantion; imipenem/cilastin;
quinolones; systemic antibodies; rifampin; polygenes; sulfunamides;
trimethoprim; glycopeptides; vancomycin; teicoplanin and
imidazoles; anti-cancer agents; anti-kaposi's sarcoma;
anti-convulsants; mephenytoin; phenobarbital; trimethadione;
anti-emetics; triethylperazine; antihistamines; chlorophinazine;
dimenhydrinate; diphenhydramine; perphenazine; tripelennamine and
the like; anti-inflammatory agents; hormonal agents;
hydrocortisone; prednisolone; prednisone; non-hormonal agents;
allopurinol; for claims water-soluble hormone drugs; antibiotics;
antitumor agents; antipyretics; analgesics; expectorants;
sedatives; muscle relaxants; antiepileptics; anticulcer agents;
antidepressants; antiallergic drugs; cardiotonics; antiarrhythmic
drugs; vasodilators; antihypertensives; diuretics; anticoagulants;
and antinarcotics; in the molecular weight range of 100-100,000
daltons; indomethacin; phenylbutazone; prostaglandins; cytotoxic
drugs; thiotepa; chloramucil; cyclophosphamide; melphala; nitrogen
mustard; methotrexate; antigens; proteins; glycoproteins; synthetic
peptides; carbohydrates; synthetic polysaccharides; lipids;
glycolipids; lipopolysaccharides(LPS); synthetic
lipopolysaccharides and with or without attached adjuvants of
synthetic muramyl dipeptide; antigens of such microorganisms as
Neisseria gonorrhea; Mycobacterium tuberculosis; Picarinii
Pnfumonia; Herpes virus (humonis types 1 and 2); Herpes zoster;
Candidia albicans; Candida tropicalis; Trichomonas vaginalis;
Haemophitus vaginalis; Group B streptoccoccus ecoli; Microplasma
hominis; Hemophilus ducreyi; Granuloma inguimale; Lymphopathia
venerum; Treponema palidum; Brucela aborus Brucela meitensis
Brucela suis; Brucella canis Campylobacter fetus; Campylobacer
fetus intesinalis; Leptospira pomona; Listeria monocytogenes;
Brucella ovis; Equine herpes virus 1; Equine arteritis virus;
IBR-IBP virus; Chlamydia psittaci; Trichomonas foetus; Taxoplasma
gondii; Escherichia coli; Actinobacillus equili; Salmonella abortus
ovis; Salmonella abortus eui; Pseudomonas aeruginosa;
Corynebacterium equi; Corynebacterium pyogenes; Actinobaccilus
seminis; Mycoplasma bovigenitalium; Aspergilus fumigatus; Absidia
ramosa; Trypanosoma equiperdum; Babesia cabali; Clostridium tetani;
antibodies which counteract the above microorganisms; and enzymes
including ribonuclease; neuramidinase; trypsin; glycogen
phosphorylase; sperm lactic dehydrogenase; sperm hyaluronidase;
adenossinetriphosphase; alkaline phosphatase; alkaline phospha
esterase; amino peptides; typsin chymotrypsin amylase; muramidase;
acrosornal proteinase; diesterase; glutamic acid dehydrogense;
succunic and dehydrogenase; beta-glycophosphatase lipase; ATP-ase
alpha-peptate gamma-glutamyiotranspeptidase;
sterold-beta-ol-dehydrogenase; DPN-di-aprorase; and combinations
thereof.
6. The composition of claim 5 wherein the agent is selected from
the group consisting of antibacterial agents; antibacterial
peptides; antimycobacterial agents; antimycotic agents; antiviral
agents; antiparasitic agents; antifungal; hormonal peptides;
cardiovascular agents; narcotic antagonist; analgesics;
anesthetics; vaccines; insulins; HIV therapeutic drugs (protease
inhibitors); estrogens; progestins; gastrointestinal therapeutic
agents; non-steroidal anti-inflammatory agents; parasympathoimetic
agents; psychotherapeutic agents; tranquilizers; decongestants;
sedative-hypnotics; non-estrogenic and non-progestional steroids;
sympathomimetic agents; vaccines; vitamins; nutrients;
anti-malarial compounds; anti-migraine drugs; electrolyte
replacements; ergot alkaloids; analgetics; non-narcotics;
anti-cancer agents; anticonvulsants; anti-emetics; antihistamines;
anti-inflammary agents; prostaglandins; cytotoxic drugs; antigens;
antibodies; enzymes; growth factors; immunomodulators; pheromones;
prodrugs; psychotropic drugs; appetite suppresants/stimulants; and
combinations thereof.
7. The composition of claim 5 wherein the biologically active agent
is a peptide or polypeptide.
8. The composition of claim 7 wherein the biologically active agent
is a polypeptide.
9. The composition of claim 8 wherein the molecular weight of the
polypeptide is between 1,000-250,000 daltons.
10. The composition of claim 9 wherein the polypeptide is histatin
consisting of 12 amino acids and having a molecular weight of 1563
daltons.
11. The composition of claim 9 having analogs of histatin with
chain lengths of from 11-24 amino acids of molecular weights from
1,500-3,000 daltons and characterized by the following
structures:
1. D S H A K R H H G Y K R K F H E K H H S K R G Y
2. K R H H G Y K R K F H E K H H S H R G Y R
3. K R H H G Y K R K F H E K H H S R
4. R K F H E K H H S H R G Y R
5. A K R H H G Y K R K F H
6. *A K R H H G Y K R K F H
7. K R H H G Y K R K F
*D-amino acid.
12. The composition of claim 1 wherein release profiles of variable
rates or durations are achieved by blending uncapped and capped
poly(lactide/glycolide) as a cocktail in variable amounts.
13. The composition of claim 1 wherein release of profiles of
variable rates or duration are achieved by blending uncapped and
capped polymer in different ratios within the same
poly(lactide/glycolide).
14. The composition of claim 1 wherein said biodegradable
poly(lactide/glycolide) is in an oil phase, and is present in about
1-50% (w/w).
15. The composition of claim 14 wherein a concentration of the
active agent is in the range of 0.1 to about 60% (w/w).
16. The composition of claim 15 wherein a ratio of the inner
aqueous to oil phases is about 1/4 to 1/40 (v/v).
17. The composition of claim 8 when the polypeptide is histatin and
is inactive at a low pH, a pH-stabilizing agent of inorganic salts
is added to an inner aqueous phase to maintain biological activity
of the released peptide.
18. The composition of claim 8 wherein when the polypeptide is
histatin and is inactive at a low pH, a non-ionic surfactant
selected from polyoxyethylene sorbitan fatty acid esters and
polyoxyethylene-polyoxypropylene block copolymers is added to an
inner aqueous phase to maintain biological activity of the released
polypeptide.
19. The composition of claim 17 wherein placebo spheres loaded with
the pH-stabilizing agents are coadministered with
polypeptide-loaded spheres to maintain the solution pH around the
microcapsules and preserve the biological activity of the released
peptide in instances where the addition of pH-stablizing agents in
the inner aqueous phase is undesirable for the successful
encapsulation of the acid pH sensitive polypeptide.
20. The composition of claim 18 wherein placebo spheres loaded with
non-ionic surfactant are coadministered with polypeptide-loaded
spheres to maintain biological activity of the released peptide
where the addition of non-ionic surfactants in the inner aqueous
phase is undesirable for successful encapsulation of the acid pH
sensitive polypeptide.
21. The composition of claim 8 wherein the polypeptide is
histatin.
22. The composition of claim 21, comprising a capacity to
completely release histatin in an aqueous physiological environment
within from 1 to 40 days with a 100/0 blend of uncapped and
end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to
52/48, and a molecular weight less than 15,000 daltons.
23. The composition of claim 22 wherein the histatin iscompletely
released within 18 to 40 days and the molecular weight of the
poly(lactide/glycolide) is within the range of 28,000 to 40,000
daltons.
24. The composition of claim 21 comprising a capacity to release up
to 90% of the histatin in an aqueous physiological environment from
28-70 days with a 1/99 blend of uncapped and end-capped
poly(lactide/glycolide) having a L/G ratio of 48/52 to 52/48 and a
molecular weight range of 10,000-40,000 daltons.
25. The composition of claim 21 comprising a capacity to release up
to 80% of histatin in an aqueous physiological environment from
56-100 days with a 1/99 blend of uncapped and end-capped
poly(lactide/glycolide) having a L/G ratio of 75/25 and a molecular
weight of less than 15,000 daltons.
Description
III. FIELD OF THE INVENTION
This invention relates to compositions comprising active core
material(s) such as biologically active agent(s), drug(s) or
substance(s) encapsulated within an end-capped or a blend of
uncapped and end-capped biodegradable-biocompatable
poly(lactide/glycolide) polymeric matrix useful for the effective
prevention or treatment of bacterial, viral, fungal, or parasitic
infections, and combinations thereof. In the areas of general and
orthopedic surgery, and the treatment of patients with infectious
or chronic disease conditions, this invention will be especially
useful to physicians, dentists and veternarians.
IV. BACKGROUND OF THE INVENTION
Wounds characterized by the presence of infection, devitalized
tissue, and foreign-body contaminants have high infection rates and
are difficult to treat.
To prevent infection, in bone and soft tissue systemic antibiotics
must be administered within 4 hours after wounding when circulation
is optimal. This has been discussed by J. F. Burke in the article
entitled "The Effective Period of Preventive Antibiotic Action in
Experimental Incisions and Dermal Lesions", Surgery, Vol. 50, Page
161 (1961). If treatment of bacterial infections is delayed, a
milieu for bacterial growth develops which results in complications
associated with established infections. (G. Rodeheaver et al.,
"Proteolytic Enzymes as Adjuncts to Antibiotic Prophylaxis of
Surgical Wounds", American Journal of Surgery, Vol. 127, Page 564
(1974)). Once infections are established it becomes difficult to
systemically administer certain antibiotics for extended periods at
levels that are safe and effective at the wound site. Unless
administered locally, drugs are distributed throughout the body,
and the amount of drug hitting its target is only a small part of
the total dose. This ineffective use of the drug is compounded in
the trauma patient by hypovolemic shock, which results in a
decreased vascular flow to tissues. (L. E. Gelin et al., "Trauma
Workshop Report:Schockrheology and Oxygen Transport", Journal
Trauma, Vol. 10, Page 1078 (1970)).
Additionally, infections caused by multiple-antibiotic resistant
bacterial are on the upswing and we are on the verge of a potential
world-wide medical disaster. According to the Centers for Disease
Control, 13,300 patients died in U.S. hospitals in 1992 from
infections caused by antibiotic-resistant bacteria.
Methicillin-resistant S. aureus (MRSA) is rapidly emerging as the
"pathogen of the 90's":
a. Some major teaching hospitals in U.S. report that up to 40% of
strains of S. aureus isolated from patients are resistant to
methicillin. Many of these MRSA strains are susceptible only to a
single antibiotic (vancomycin).
b. Should MRSA also develop resistance to vancomycin, the mortality
rate among patients who develop MRSA infections could approach 80%,
thereby increasing the threat of this infectious killer.
Moreover, Vancomycin resistance is on the up-swing:
a. 20% of Enterococci are now resistant to vancomycin
b. In 1989, only one hospital in New York City reported
vancomycin-resistant Enterococci. By 1991, the number of hospitals
reporting vancomycin resistance rose to 38.
c. transfer of vancomycin-resistant gene (via plasmid) has been
shown experimentally between Enterococcus and S. aureus.
Many major pharmaceutical companies around the world have either
completely eliminated or significantly reduced their research and
development programs in the area of antibiotic research. According
to a 1994 report by the Rockefeller University Workshop in Multiple
Antibiotic Resistant Bacteria, we are on the verge of a "medical
disaster that would return physicians back to the pre-penicillin
days when even small infections could turn lethal due to the lack
of effective drugs."
Despite recent advances in antimicrobial therapy and improved
surgical techniques, osteomyelitis (hard tissue or bone infection)
is still a source of morbidity often necessitating lengthy
hospitalization. The failure of patients with chronic osteomyelitis
to respond uniformly to conventional treatment has prompted the
search for more effective treatment modalities. Local antibiotic
therapy with gentamicin-impregnated poly(methylmethacrylate) (PMMA)
bead chains (SEPTOPAL TM, E. Merck, West Germany) has been utilized
in Germany for the treatment of osteomyelitis for the past decade
and has been reported to be efficacious in several clinical
studies. The beads are implanted into the bone at the time of
surgical intervention where they provide significantly higher
concentrations of gentamicin than could otherwise be achieved via
systemic administration. Serum gentamicin levels, on the other
hand, remain extremely low thereby significantly reducing the
potential for nephro- and ototoxicity that occurs in some patients
receiving gentamicin systemically.
Since SEPTOPAL TM is not currently approved by the Food and Drug
Administration for use in the United States, some orthopedic
surgeons in this country are fabricating their own "physician-made
beads" for the treatment of chronic osteomyelitis. A major
disadvantage of the beads, however, is that because the PMMA is not
biodegradable it represents a foreign body and should be removed at
about 2-weeks postimplantation thereby necessitating in some cases
an additional surgical procedure. A biodegradable-biocompitable,
antibiotic carrier, on the other hand, would eliminate the need for
this additional surgical procedure and may potentially reduce both
the duration as well as the cost of hospitalization.
The concept of local, sustained release of antibiotics into
infected bone is described in recent literature wherein
antibiotic-impregnated PMMA macrobeads are used to treat chronic
osteomyelitis. The technique as currently used involves mixing
gentamicin with methylmethacrylate bone cement and molding the
mixture into beads that are 7 mm in diameter. These beads are then
locally implanted in the infected site at the time of surgical
debridement to serve as treatment. There are, however, significant
problems with this method. These include: 1) initially, large
amounts of antibiotics diffuse from the cement but with time the
amount of antibiotic leaving the cement gradually decreases to
subtherapeutic levels; 2) the bioactivity of the antibiotic
gradually decreases; 3) methylmethacrylate has been shown to
decrease the ability of polymorphonuclear leukocytes to phagocytize
and kill bacteria; 4) the beads do not biodegrade and usually must
be surgically removed; and 5) the exothermic reaction that occurs
during curing of methymethacrylate limits the method to the
incorporation of only thermostable antibiotics (primarily
aminoglycosides). Nevertheless, preliminary clinical trials using
these beads indicate that they are equivalent in efficacy to longer
term (4-6 weeks) administration of systemic antibiotics.
In many instances, infectious agents have their first contact with
the host at a mucosal surface; therefore, mucosal protective immune
mechanisms are of primary importance in preventing these agents
from colonizing or penetrating the mucosal surface. Numerous
studies have demonstrated that a protective mucosal immune response
can best be initiated by introduction of the antigen at the mucosal
surface, and parenteral immunization is not an effective method to
induce mucosal immunity. Antigen taken up by the gut-associated
lymphoid tissue (GALT), primarily by the Peyer's patches in mice,
stimulates T helper cell (Th) to assist in IgA B cell responses or
stimulates T suppressor cells (Ts) to mediate the unresponsiveness
of oral tolerance. Particulate antigen appears to shift the
response towards the (Th) whereas soluble antigens favor a response
by the (Ts). Although studies have demonstrated that oral
immunization does induce an intestinal mucosal immune response,
large doses of antigen are usually required to achieve sufficient
local concentrations in the Peyer's patches. Unprotected protein
antigens may be degraded or may complex with secretory IgA in the
intestinal lumen.
In the process of vaccination, medical science uses the body's
innate ability to protect itself against invading agents by
immunizing the body with antigens that will not cause the disease
but will stimulate the formation of antibodies that will protect
againts the disease. For example, dead organisms are injected to
protect against bacterial diseases such as typhoid fever and
whooping cough, toxins are injected to protect against viral
diseases such as poliomyelitis and measles.
It is not always possible, however, to stimulate antibody formation
merely by injecting the foreign agent. The vaccine preparation must
be immunogenic, that is, it must be able to induce an immune
response. Certain agents such as tetanus toxoid are innately
immunogenic, and may be administered in vaccines without
modification. Other important agents are not immunogenic, however,
and must be converted into immunogenic molecules before they can
induce an immune response.
The immune response is a complex series of reactions that can
generally be described as follows:
1. the antigen enters the body and encounters antigen-presenting
cells which process the antigen and retain fragments of the antigen
on their surfaces;
2. the antigen fragment retained on the antigen presenting cells
are recognized by T cells that provide help to B cells; and
3. the B cells are stimulated to proliferate and divide into
antibody forming cells that secrete antibody against the
antigen.
Most antigens only elicit antibodies with assistance from the T
cells and, hence, are known as T-dependent (TD). These antigens,
such as proteins, can be processed by antigen presenting cells and
thus activate T cells in the process described above. Examples of
such T-dependent antigens are tetanus and diphtheria toxoids.
Some antigens, such as polysaccharides, cannot be properly
processed by antigen presenting cells and are not recognized by T
cells. These antigens do not require T cell assistance to elicit
antibody formation but can activate B cells directly and, hence,
are known as T-independent antigens (TI). Such T-independent
antigens include H.influenzae type by polyribosyl-ribitol-phosphate
and pneumococcal capsular polysaccharides.
T-dependent antigens vary from T-independent antigens in a number
of ways. Most notably, the antigens vary in their need for an
adjuvant, a compound that will nonspecifically enhance the immune
response. The vast majority of soluble T-dependent antigens elicit
only low level antibody responses unless they are administered with
an adjuvant. It is for this reason that the standard DPT vaccine
(diptheria, pertussis, tetanus) is administered with the adjuvant
alum. Insolubilization of TD antigens into an aggregated form can
also enhance their immunogenicity, even in the absence of an
adjuvant. Golub E S and W O Weigle, J. Immunol. 102:389, 1969). In
contrast, T-independent antigens can stimulate antibody responses
when administered in the absence of an adjuvant, but the response
is generally of lower magnitude and shorter duration.
Four other differences between T-independent and T-dependent
antigens are:
a) T-dependent antigens can prime an immune response so that a
memory response can be elicited upon secondary challenge with the
same antigen. Memory or secondary responses are stimulated very
rapidly and attain significantly higher titers of antibody that are
seen in primary responses. T-independent antigens are unable to
prime the immune system for secondary responsiveness.
b) The affinity of the antibody for antigen increases with time
after immunization with T-dependent but not T-independent
antigens.
c) T-dependent antigens stimulate an immature or neonatal immune
system more effectively than T-independent antigens.
d) T-dependent antigens usually stimulate IgM, IgGI, IgG2a, and IgE
antibodies, while T-independent antigens stimulate IgM, IgGI,
IgG2b, and IgG3 antibodies.
These characteristics of T-dependent vs. T-independent antigens
provide both distinct advantages and disadvantages in their use as
effective vaccines. T-dependent antigens can stimulate primary and
secondary responses which are long-lived in both adult and in
neonatal immune systems, but must frequently be administered with
adjuvants. Thus, vaccines have been prepared using only an antigen,
such as diphtheria or tetanus toxoid, but such vaccines may require
the use of adjuvants, such as alum for stimulating optima
responses. Adjuvants are often associated with toxicity and have
been shown to nonspecifically stimulate the immune system, thus
inducing antibodies of specificities that may be undesirable.
Another disadvantage associated with T-dependent antigens is that
very small proteins such as peptides, are rarely immunogenic, even
when administered with adjuvants. This is especially unfortunate
because many synthetic peptides are available today that have been
carefully synthesized to represent the primary antigenic
determinants of various pathogens, and would otherwise make very
specific and highly effective vaccines.
In contrast, T-independent antigens, such as polysaccharides, are
able to stimulate immune responses in the absence of adjuvants.
Unfortunately, however, such T-independent antigens cannot
stimulate high level or prolonged antibody responses. An even
greater disadvantage is their inability to stimulate an immature or
B cell defective immune system (Mond J. J., Immunological Reviews
64:99, 1982) Mosier D E, et al., J. Immunol. 119:1874, 1977). Thus,
the immune response to both T-independent and T-dependent antigens
is not satisfactory for many applications.
With respect to T-independent antigens, it is critical to provide
protective immunity against such antigens to children, especially
against polysaccharides such as H. influenzae and S. pneumoniae.
With respect to T-dependent antigens, it is critical to develop
vaccines based on synthetic peptides that represent the primary
antigenic determinants of various pathogens.
One approach to enhance the immune response to T-independent
antigens involves conjugating polysaccharides such H. influenzae
PRP (Cruse J. M., Lewis R. E. Jr. ed., Conjugate vaccines in
Contributions to Microbiology and Immunology, vol. 10, 1989) or
oligosaccharide antigens (Anderson P W, et al., J. Immunol.
142:2464, 1989) to a single T-dependent antigen such as tetanus or
diphtheria toxoid. Recruitment of T cell help in this way has been
shown to provide enhanced immunity to many infants that have been
immunized. Unfortunately, only low level antibody titers are
elicited, and only some infants respond to initial immunizations.
Thus, several immunizationa are required and protective immunity is
often delayed for months. Moreover, multiple visits to receive
immunizations may also be difficult for families that live distant
from medical facilities (especially in underdeveloped countries).
Finally, babies less than 2 months of age may mount little or no
antibody response even after repeated immunization.
One possible approach to overcoming these problems is to
homogeneously disperse the antigen of interest within the polymeric
matrix of appropriately sized biodegradable-biocompatible
microspheres that are specifically taken up by GALT. Eldridge et
al. have used a murine model to show that orally-administered 1-10
micrometer microspheres consisting of polymerized lactide and
glycolide, (the same materials used in resorable sutures), were
readily taken up into Peyer's patches, and the 1-5 micrometer size
were rapidly phagocytized by macrophages. Microspheres that were
5-10 micrometers (microns) remained in the Peyer's patch for up to
35 days, whereas those less than 5 micrometer disseminated to the
mesenteric lymph node (MLN) and spleen within migrating MAC-1+
cells. Moreover, the levels of specific serum and secretory
antibody to staphylococcal enterotoxin B toxoid and inactivated
influenza A virus were enhanced and remained elevated longer in
animals which were immunized orally with microencapsulated antigen
as compared to animals which received equal doses of
non-encapsulated antigen. These data indicate that
microencapsulation of an antigen given orally may enhance the
mucosal immune response against enteric pathogens. AF/R1 pili
mediate the species-specific binding of E. coli RDEC-1 with mucosal
glycoproteins in the small intestine of rabbits and are therefore
an important virulence factor. Although AF/R1 pili are not
essential for E. coli RDEC-1 to produce enteropathogenic disease,
expression of AF/R1 to produce enteropathogenic disease, expression
of AF/R1 promotes a more severe disease. Anti-AF/R1 antibodies have
been shown to inhibit the attachment of RDEC-1 to the intestinal
mucosa and prevent RDEC-1 disease in rabbits. The amino acid
sequence of the AF/R1 pilin subunit has recently been determined,
but specific antigenic determinants within AF/R1 have not been
identified.
In the current study we have used these theortical criteria to
predict probable T or B cell epitopes from the amino acid sequence
of AF/R1. Four different 16 amino acid peptides that include the
predicted epitopes have been synthesized: AF/R1 40-55 as a B cell
epitope, 79-94 as a T cell epitope, 108-123 as a T and B cell
epitope, and AF/R1 40-47/79-86 as a hybrid of the first eight amino
acids from the predicted B cell epitope and the T cell epitope. We
have used these peptides as well as the native protein to stimulate
the in vitro proliferation of lymphocytes taken from the Peyer's
patch, MLN, and spleen of rabbits which have received introduodenal
priming with microencapsulated or non-encapsulated AF/R1. Our
results demonstrate the microencapsulation of AF/R1 potentiates the
cellular immune response at the level of the Peyer's patch, thus
enhancing in vitro lymphocyte proliferation to both the native
protein and its linear peptide antigens. CFA/I pili, rigid
thread-like structures which are composed of repeating pilin
subunits of 147 amino acid found on serogroups 015, 025, 078, and
0128 of enterotoxigenic E. coli (ETEC) (1-4, 18). CFA/I promotes
mannose resistant attachment to human brush borders (5); therefore,
a vaccine that established immunity against this protein may
prevent the attachment to host tissues and subsequent disease. In
addition, because the CFA/I subunit shares N-terminal amino acid
sequence homology with CS1, CFA/II (CS2) and CFA/IV (CS4) (4), a
subunit vaccine which contained epitopes from this area of the
molecule may protect against infection with various ETEC.
Until recently, experiments to identify these epitopes were time
consuming and costly; however, technology is now available which
allows one to simultaneously identify all the T cell and B cell
epitopes in the protein of interest. Multiple Peptide synthesis
(Pepscan) is a technique for the simultaneous synthesis of hundreds
of peptides on polyethylene rods (6). We have used this method to
synthesize all the 140 possible overlapping actapeptides of the
CFA/I protein. The peptides, still on the rods, can be used
directly in ELISA assays to map B call epitopes (6. 12-14). We have
also synthesized all the 138 possible overlapping decapeptides of
the CFA/I protein. For analysis of T cell epitopes, these peptides
can be cleaved from the rods and used in proliferation assays (15).
Thus this technology allows efficient mapping and localization of
both B cell and T cell epitopes to a resolution of a single amino
acid (16). These studies were designed to identify antigenic
epitopes of ETEC which may be employed in the construction of an
effective subunit vaccine.
CFA/I pili consist of repeating pilin protein subunits found on
several serogroups of enterotoxigenic E coli (ETEC) which promote
attachment to human intestinal mucosa. We wished to identify areas
within the CFA/I molecule that contain imunodominant T cell
epitopes that are capable of stimulating the cell-mediated portion
of the immune response in primates as well as immunodominant B cell
epitopes. To do this, we (a) resolved the discrepancy in the
literature on the complete amino acid sequence of CFA/I, (b)
immunized three Rhesus monkeys with multiple i.m. injections of
purified CFA/I subunit in Freund's adjuvant, (c) synthesized 138
overlapping decapeptides which represented the entire CFA/I protein
using the Pepscan technique (Cambridge Research Biochemicals), (d)
tested each of the peptides for their ability to stimulate the
spleen cells from the immunized monkeys in a proliferative assay
(e) synthesized 140 overlapping octapeptides which represented the
entire CFA/I protein, and (f) tested serum from each monkey for its
ability to recognize the octapeptides in a modified ELISA assay. A
total of 39 different CFA/I decapeptides supported a significant
proliferative response with the majority of the responses occurring
within distinct regions of the protein (peptides beginning with
residues 8-40, 70-80, and 126-137). Nineteen of the responsive
peptides contained a serine residue at positions 2, 3, or 4 in the
peptide, and a nine contained a serine specifically at position 3.
Most were predicted to be configured as an alpha holix and have a
high amphipathic index. Eight B cell epitopes were identified at
positions 3-11, 11-21, 22-29, 32-40, 38-45, 66-74, 93-101, and
124-136. The epitope at position 11-21 was strongly recognized by
all three individual monkeys, while the epitopes at 93-101,
124-136, 66-74, and 22-29 were recognized by two of the three
monkeys.
Recent advances in the understanding of B cell and T cell epitopes
have improved the ability to select probably linear epitopes from
the amino acid sequence using theoretical criteria. B cell epitopes
are often composed of a string of hydrophilic amino acids with a
high flexibility index and a high probability of turns within the
peptide structure. Prediction of T cell epitopes are based on the
Rothbard method which identifies common sequence patterns that are
common to known T cell epitopes or the method of Berzofsky and
others which uses a correlation between algorithms predicting
amphipathic helices and T cell epitopes.
V. SUMMARY OF THE INVENTION
This invention relates to active core materials such as
biologically active agent(s), drug(s), or substance(s) encapsulated
within a biodegradable-biocompatable polymeric matrix. In view of
the enormous scope of this invention it will be presented herein as
Phases I, II, and III. Phase I illustrates the encapsulation of
antibiotics within a biodegradable-biocompatable polymeric matrix
for the prevention and treatment of wound infections. Phase II
illustrates the encapsulation of antigens (more specifically,
oral-intestinal vaccine antigens) within a
biodegradable-biocompatable polymeric matrix against diseases such
as those caused by enteropathogenic organism. Phase III illustrates
the use of a biodegradable-biocompatible polymeric matrix for
burst-free programmable sustained release of biologically active
agents, inclusive of peptides, over a period of up to 100 days in
an aqueous physiological environment.
Controlled drug delivery from a biodegradable-biocompatable matrix
offers profound advantages over conventional drug/antigen dosing.
Drugs/antigens can be used more effectively and efficiently, less
drug/antigen is required for optimal therapeutic effect and, in the
case of drugs, toxic side effects can be significantly, reduced or
essentially eliminated through drug targeting. The stability of
some drugs/antigens can be improved allowing for a longer
shelf-life, and drugs/antigens with a short half-life can be
protected within the matrix from destruction, thereby ensuring
sustained release of active agent over time. The benefit of a
continuous sustained release of drug/antigen is beneficial because
drug levels can be maintained within a constant therapeutic range
and antigen can be presented either continuously or in a pulsatile
mode as required to stimulate the optimal immune response. All of
this can be accomplished with a single dose of encapsulated
drug/antigen.
This invention contemplates, but is not limited to, medically
acceptable methods for the effective local delivery of biologically
active agents that, of themselves, are directly (e.g. drugs, such
as antibiotics) or indirectly (e.g. vaccine antigens) therapeutic
or prophylactic. It also includes drugs/agents that elicit/modulate
natural biological activity.
Wounds characterized by the presence of infection, devitalized
tissue, and foreign-body contaminants have high infection rates and
are difficult to treat. This invention describes antibiotic
formulation encapsulated within microspheres of a
biodegradable-biocompatable polymer that, when applied locally to
contaminated or infected wounds, provides immediate, direct, and
sustained (over a period up to 100 days), high concentrations of
antibiotic in the wound site (soft tissue and bone). By
encapsulating antibiotics and applying them directly, one can
achieve a significant reduction in nonspecific binding of the drug
to body proteins, a phenomena commonly observed following
conventional systemic administration of free drugs. Thus, less drug
is required, higher concentrations are maintained at the site of
need, and efficacy is enhanced. This approach provides superior
treatment over conventional systemic administration of antibiotics
for wound infections because higher bacteriocidal concentrations
can be achieved and maintained in the wound environment. Higher
concentrations kill more bacteria. Applicants' invention for this
application is described in Phase I. Furthermore, applicants
reasoned that a protective mucosal immune response might be best
initiated by introduction of an antigen at the mucosal surface,
because unprotected protein antigens delivered in a free form may
be degraded or may complex with secretory IgA in the intestinal
lumen precluding entry and subsequent processing in local immune
cells. The formulation of microspheres containing antigen small
enough in size to be phagocytized locally in the gut was envisioned
as being able to induce an elevated localized immune response.
Applicants' invention for this application is described in Phase
II. In summary, applicants propose using several methods for the
local application of drugs including: 1) the direct application of
the encapsulated drug to a surgical/traumatized area, 2) oral
delivery that provides either local deposition of microencapsulated
antigen/drugs at mucosal membranes or transport across these
membranes to provide local adherence of microencapsulated
drugs/antigen to mucosal membranes to provide sustained release of
drug/antigen into soft tissue or a body cavity, and/or 3) sustained
intercellular or extracellular drug/antigen release following
subcutaneous injection.
In those instances where antibiotics are administered locally,
applicants have found that the controlled release of the antibiotic
from within a biodegradable-biocompatable polymeric matrix within
14 days to about 4 weeks without significant drug trailing is
especially useful. However, if desired, the release of a
biologically active agent from a polymeric matrix comprised of an
active agent and a blend of uncapped and end-capped biodegradable
poly DL(lactid-co-glycolide), can be controlled over a period of 1
to about 100 days without significant drug dumping or trailing.
Such novel biocompatible-biodegradable microspheres developed with
a burst-free programmable sustained release of biologically active
agents, inclusive of polypeptides, are described in applicants'
U.S. patent application Ser. No. 08/590,973 filed Jan. 24,
1996.
When antibiotics are administered systemically in the conventional
manner, or locally as contemplated by the applicants, the immune
response to the antibiotic and the potential for hypersensitivity
and/or anaphylactoid response (especially to beta-lactam
antibiotics such as penicillins/ampicillin) is a clinical concern.
In early studies the inventors observed a specific IgG response to
ampicillin as it was released from the microencapsulated
formulation (illustrated in the histogram, FIGS. 1 and 2). This
response is reminiscent of antibody elicited by vaccine antigens in
conventional vaccines. The response to vaccine antigens is known to
be accentuated by the use of an adjuvant such as alum. Alum is a
crude, less adaptable delivery vehicle than its counterpart, the
biodegradable-biocompatable poly DL(lactide-co-glycolide), of this
invention--the polymeric matrix. This knowledge stimulated
additional studies relevant to the effects of sustain release of
agents on the immune response.
There are, in general, two forms of localized delivery which can be
achieved with PLGA microspheres-delivery which is localized to
individual cells of the body (intracellular delivery); and delivery
which is localized to tissues within a specific region of the body
(localized extracellular delivery).
Applicants have prepared antibiotic and hepatitis vaccine
formulations which functioned by delivering localized extracellular
doses of their active agents. This was achieved by using relatively
large microspheres which served as a depot for the drug or antigen.
Their large size 40-100 microns in diameter precluded their being
phagocytized or diffusing throughout the intercellular fluid
compartments of the body. Their drug agent loads were thus released
within their immediate vicinity which resulted in the generation of
very high local concentrations of antibiotic or the release of
sufficiently high concentrations of free antigen to induce an
immune response.
The large-diameter antibiotic bearing microspheres were originally
developed by applicants primarily for topical application on
exposed debrided tissues of combat wounds. However, an inherent
property exhibited by the antibiotics when topically applied to a
wound site is the generation of measurable levels of immune
response. This concept of local delivery by topical application of
microspheres to tissue to achieve localized concentrations of
therapeutic agents was subsequently applied to the development of
an oral vaccine for protection against traveler's diarrhea caused
by E. coli. Vaccine antigen was encapsulated into microspheres
whose diameters were predominantly in the 5-10 micron size range
based on an understanding that microspheres of this size would not
readily be either phagocytied or transported across the gut wall
into the body. Ingestion of these microspheres thus constituted a
localed delivery achieved by topical application of the spheres to
the wall tissue of the gut. This topical application resulted in
the localized trapping of a small percentage of these sphere into
the Peyer's patches where the spheres proceeded to release their
antigen in a localized fashion to immune cells located within the
intestinal Patches.
The concept of localized sustained local delivery has been further
extended to the delivery of analgesics and anesthetics to exposed
dental pulp to control pain and inflammatory responses. Again, the
PLGA microsphere used for this type of delivery are relatively
large (40-100 um in diameter) and serve as a topical depot for
localized extracellular release of the drug.
Consistent with their understanding of the inherent immunogenic
properties exhibited by active core materials in vivo, applicants
have moved on to other non-topical application methods of using
their microsphere delivery system. Some of these center on the use
of small diameter microspheres ranging from sub micron to under 5
microns in diameter. These spheres allow intracellular targeting of
drug or antigen. They also allow for transmucosal delivery of drugs
or antigens. The concept of localized delivery in these instances
refers to the localized delivery of drug or agent within individual
target cells of the body regardless of their location or
distribution within the body. This approach is useful in
development of antitubercular, antimalarial, antiviral, and
antichlamydial formulations against intracellular parasites. It is
also useful for the development of vaccines against intracellular
parasites and for direct delivery of agents to presenting cells of
the immune system.
Another nontopical application method of using PLGA microspheres
resides in their usefulness as injectable depots for drugs intended
for either localized or systemic delivery. Typically larger
diameter microspheres are used for depots as these are less likely
to diffuse away. The local or systemic nature of these delivery
systems is, in part a function of the release rate of the drug from
the depot and the diffusional and solubility characteristics of the
drug being released. Cancer chemotherapeutics, systemic
antibiotics, delivery of antibiotics to infected bone are potential
application of this system. Additional this non-topical systemic
depot application can be extended to the iv injection of
cancer-agent laden microspheres to embolize and destroy a malignant
tumor. Additionally, the PLGA microspheres can be used as a carrier
to deliversubstances useful for the in modification of cells or
genes in bioengineering or genetic procedures.
Interest in the concept that antigens encapsulated within a
biodegradable-biocompatible polymeric matrix could be formulated as
a vaccine with superior efficacy over conventional vaccines,
originated from the inventors' own observations that the drug,
ampicillin, when sustain released from poly
DL(lactide-co-glycolide) elicited antibody production. In these
studies, the applicants were able to measure specific IgG
antibodies to free ampicillin and to ampicillin released from
microencapsulated ampicillin formulations in the sera of mice
previously "treated" with the ampicillin formulations using ELISA.
Numerous other studies also document the ability of beta-lactam
antibiotic to elicit antibody. Selected, more recent studies whose
findings are consistent with earlier discoveries made by applicants
when conducting experiements with ampicillin include those by Klein
et al. (1993) who detected specific IgG antibodies (IgG and IgG3
subclasses) to the B-lactam ring in patients receiving penicillin
therapy, work by Nagakura et al. (1990) which detected specific
antibodies to cephalexin, a B-lactam antibiotic in the sera of
guinea pigs, and Auci et al. (1993) who detected benzyl penicilloyl
specific IgM, IgG IgE, and IgA antibody forming cells in lymphoid
cells of mice given benzyl penicilloyl-Keyhole Limpet Hemocyanin.
Pharmaceutical compositions of antigens encapsulated with poly
DL(lactide-co-glycolide) are described in Phase II. The
microspheres of the invention allow for introduction of vaccine
antigens to mucosal surfaces in particles that can be subsequently
taken up locally by phagocytic cells. Such an approach for both
drugs and antigens provides significant advantages in potency and
efficacy over conventional systemically administered drugs or
vaccines. A partial list of biologically active agents or drugs
that will potentially derive significant medical benefits from this
delivery system includes: antibacterial agents; peptides;
polypeptides; antibacterial peptides; antimycobacterial agents;
antimycotic agents; antiviral agents; antiparastic agents;,
antifungal; antiyeast agents; hormonal peptides; cardiovascular
agents; hormonal peptides; cardiovascular agents; narcotic
antagonists; analgesics; anesthetics; insulins; steroids including
HIV therapeutic drugs (including protease inhibitors) and AZT;
estrogens; progestins; gastrointestinal therapeutic agents;
non-steroidal anti-inflammatory agents; parasympathoimetic agents;
psychotherapeutic agents; tranquilizers; decongestants;
sedative-hypnotics; non-estrogenic and non-progestional steroids;
sympathomimetic agents; vaccines; vitamins; nutrients;
anti-migraine drugs; electrolyte replacements; ergot alkaloids;
anfi-inflammary agents; prostaglandins; cytotoxic drugs; antigens;
antibodies; enzymes; growth factors; immunomodulators; pheromones;
prodrugs; psychotropic drugs; nicotine; antiblood clotting drugs;
appetite suppressants/stimulants and combinations thereof;
contraceptive agents include estrogens such as diethyl silbestrol;
17-beta-estradiol; estrone; ethinyl estradiol; mestranol;
progestins such as norethindrone; norgestryl; ethynodiol diacetate;
lynestrenol; medroxyprogesterone acetate; dimethisterone; megestrol
acetate; chlormadinone acetate; norgestimate; norethisterone;
ethisterone; melentate; norgestimate; norethisterone; ethisterone;
melengestrol; norethynodrel; and spermicidal compounds such as
nonyphenoxypolyoxyethylene glycol; benzethonium chloride;
chlorindanol; include gastrointestinal therapeutic agents such as
aluminum hydroxide; calcium carbonate; magnesium carbonate; sodium
carbonate and the like; non-steroidal antifertility agents;
parasympathomimetic agents; psychotherapeutic agents; major
tranquilizers such as chloropromaquine HCL; clozapine;
mesoridazine; metiapine; reserpine; thioridazine; minor
tranquilizers such as chlordiazepoxide; diazepam; meprobamate;
temazepam and the like; rhinological decongestants;
sedative-hypnotics such as codeine; phenobarbital; sodium
pentobarbital; sodium secobarbital; other steroids such as
testosterone and testosterone propionate; sulfonmides;
sympathomimetic agents; vaccines; vitamins and nutrient such as the
essential amino acids; essential fats; anti-HIV agents; including
AZT; antimalarials such as 4-aminoquinolines; 8 aminoquinolines;
pyrimethamine; anti-migraine agents such as mazindol; phentermine;
anti-Parkinson agents such as L-dopa; antispasmodics such as
atropine; methscopolamine bromide; antispasmodics and
anticholingeric agents such as bile therapy; digestants; enzymes
and the like; antitussives such as dextromethorphan and noscapine;
bronchodilators; cardiovascular agents such as anti-hypertensive
compounds; Rauwolfia alkaloids; coronary vasodilators;
nitroglycerin; organic nitrites; pentaerythriotetranitrate;
electrolyte replacements such as potassium chloride; ergotalkaloids
such as ergotamine with and without caffein; hydrogenated ergot
alkaloids; dihydroergocristine methanesulfate; dihydroergocornine
methanesulfonate; dihydroergokroyptine methaneusulfate and
combinations thereof; alkaloids such as atropine sulfate;
Belladonna; hyoscine hydrobromide; analgesics; narcotics such as
codeine; dihydrocodienone; meperidine; morphine; non-narcotics such
as salicylates; aspirin; acetaminophen; and d-propoxyphene;
antibiotics such as the cephalosporins including ceflacor and
cefuroxime; chloranphenical; gentamicin; Kanamycin A. Kanamycin B;
the penicillins; ampicillin; amoxicillin; streptomycin A; antimycin
A; chloropamtheniol; metromidazole; oxytetracycline penicillin G;
the tetracyclines; including minocycline; fluoro-quinolones
including ciprofloxacin; ofoxacin; macrolides including
clarithromycin; frythromycin; aminioglycosides including
gentamicin; amikacin; tobramycin and kanamycin; beta-lactams
including ampacillin; polymyxin-B; amphotercin-B; aztrofonam;
chloramphenicol; fusidans; lincosamides; metronidazole;
nitro-furantion; imipenem/cilastin; quinolones; systemic antibodies
including rifampin; polygenes; sulfunamides; trimethoprim;
glycopeptides including vancomycin; teicoplanin and imidazoles;
anti-cancer agents; including anti-kaposi's sarcoma;
anti-convulsants such as mephenytoin; phenobarbital; trimethadione;
anti-emetics such as triethylperazine; antihistamines such as
chlorophinazine; dimenhydrinate; diphenhydramine; perphenazine;
tripelennamine and the like; anti-inflammatory agents such as
hormonal agents; hydrocortisone; prednisolone; prednisone;
non-hormonal agents; allopurinol; for claims water-soluble hormone
drugs; antibiotics; antitumor agents; anti infalmmatory agents;
antipyretics; analgesics; antitussives; expectorants; sedatives;
muscle relaxants; antiepileptics; anticulcer agents;
antidepressants; antiallergic drugs; cardiotonics; antiarrhythmic
drugs; vasodilators; antihypertensives; diuretics; anticoagulants;
and antinarcotics; in the molecular wight range of 100-100,000
daltons; indomethacin; phenylbutazone; prostaglandins; cytotoxic
drugs such as thiotepa; chloramucil; cyclophosphamide; melphala;
nitrogen mustard; methotrexate; antigens such as proteins;
glycoproteins; synthetic peptides; carbohydrates; synthetic
polysaccharides; lipids; glycolipids; lipopolysaccharides(LPS);
synthetic lipopolysaccharides and with or without attached
adjuvants such as synthetic muramyl dipeptide derivatives; antigens
of such microorganisms as Neisseria gonorrhea; Mycobacterium
tuberculosis; Picarinii Pnfumonia; Herpes virus (humonis types 1
and 2); Herpes zoster; Candidia albicans; Candida tropicalis;
Trichomonas vaginalis; Haemophilus vaginalis; Group B
streptoccoccus ecoli; Microplasma hominis; Hemophilus ducreyi;
Granuloma inguimale; Lymphopathia venerum; Treponema palidum;
Brucela aborus Brucela meitensis Brucela suis; Brucella canis
Campylobacter fetus; Campylobacer fetus intesinalis; Leptospira
pomona. Listeria monocytogenes; Brucella ovis; Equine herpes virus
1; Equine arteritis virus; IBR-IBP virus; Chlamydia psittaci;
Trichomonas foetus; Taxoplasma gondii; Escherichia coli;
Actinobacillus equuli; Salmonella abortus ovis. Salmonella abortus
eui; Pseudomonas aeruginosa; Corynebacterium equi; Corynebacterium
pyogenes; Actinobaccilus seminis; Mycoplasma bovigenitalium;
Aspergilus fumigatus; Absidia ramosa; Trypanosoma equiperdum;
Babesia cabali; Clostridium tetani; antibodies which counteract the
above microorganisms; and enzymes such as ribonuclease;
neuramidinase; trypsin; glycogen phosphorylase; sperm lactic
dehydrogenase; sperm hyaluronidase; adenossinetriphosphase;
alkaline phosphatase; alkaline phospha esterase; amino peptides;
typsin chymotrypsin amylase; muramidase; acrosomal proteinase;
diesterase; glutamic acid dehydrogense; succunic and dehydrogenase;
beta-glycophosphatase lipase; ATP-ase alpha-peptate
gamma-glutamyiotranspeptidase; sterold-beta-ol-dehydrogenase;
DPN-di-aprorase; and combinations thereof. Having generally
described the invention; a further understanding can be obtained by
reference to certain specific examples which are provided herein
for purpose of illustration only and are not intended to be
limiting unless otherwise specified. Moreover; the polymeric matrix
of this invention may be used for the in situ production and
controlled release of products that are produced by the controlled
release of encapsulated reactants. Additionally; effective testing
or monitoring devices for chemical agents or bioactive agents can
be made by encapsulating reagents which react as they are released
from the polymeric matrix, with agents sought to be detected. The
novel delivery system of this invention is applicable to all
categories of active substances capable of being used for the
prevention and/or treatment of human, animal and plant diseases.
This delivery system is also applicable to the design of novel
diagnostic tests. Additionally, it can be useful for the delivery
to a subject of a polyfunctional mixture or cocktail formulation of
encapsulated active (i.e. biologically) substances for the
prevention and/or treatment of diseases the same or different. The
encapsulated formulation ingredients would be comprised of multiple
drugs, multipe vaccines or a combination thereof.
Applicants contemplate that the invention will be useful in the
formulation of disease specific compositions for the prevention
and/or treatment of diseases and/or ailments which include: viral
infections; bacterial infections; fungal infections; yeast
infections; parastic infections and more specific diseases and/or
ailments; such as as, aids; alzheimer's dementia; angiogenesis
diseases; aphthour ulcers in AIDS patients; asthma; atopic
dermatitis; psoriasis; basal cell carcinoma; benign prostatic
hypertrophy; blood substitute; blood substitute in surgery
patients; blood substitute in trauma patients; breast cancer;
breast cancer; cutaneous & metastatic; cachexia in AIDS;
campylobacter infection; Cancer; pnemonia; sexually transmitted
diseases (STDs); cancer; viral dieases; candida albicians in AIDS
and cancer; candidiasis in HIV infection; pain in cancer;
pancreatic cancer; parkinson's disease; peritumoral brain edema;
postoperative adhesions (prevent); proliferative diseases; prostate
cancer; ragweed allergy; renal disease; restenosis; rheumatoid
arthritis; rheumatoid arthritis; allergies; rotavirus infection;
scalp psoriasis; septic shock; small-cell lung cancer; solid
tumors; stroke; thrombosis; type I diabetes; type I diabetes
w/kidney transplants; type II diabetes; viseral leishmaniasis;
malaria; periodontal or gum disease; cardiac rthythm disorders;
central nervous system diseases; central nervous system disorders;
cervical dystonia (spasmodic torticollis); choridal
neovascularization; chronic hepatitis c, b and a; colitis
associated with antibiotics; colorectal cancer; coronary artery
thrombosis; cryptosporidiosis in AIDS; cryptosporidium parvum
diarrhea in AIDS; cystic fibrosis; cytomegalovirus disease;
depression; social phobias; panic disorder; diabetic complications;
disabetic eye disease; diarrhea associated with antibiotics;
erectile dysfunction; genital herpes; graft-vs host disease in
transplant patients; growth hormone deficiency; head and neck
cancer; head trauma; stroke; heparin neutralization after cardiac
bypass; hepatocellular carcinoma; HIV; HIV infection; huntington's
disease; CNS diseases; hypercholesterolemia; hypertension;
inflammation; inflammation and angiogensis; inflammation in
cardiopulmonary bypass; influenza; migrain head ache; interstitial
cystitis; kaposi's sarcoma; kaposi's sarcoma in AIDS; lung cancer;
melanoma; molluscum contagiosum in AIDS; multiple sclerosis;
neoplastic meningitis from solid tumors; non-small cell lung
cancer; organ transplant rejection; osteoarthritis; rheumatoid
arthritis; osteoporosis; drug addiction; shock; ovarian cancer; and
pain.
Also contemplated here are those diseases or health conditions
capable being benefitted by the list of biologically active agents
or drugs previously listed in the Summary of the Invention.
Effects of Microencapsulated Antibiotics on the Immune Response
Preclinical studies evaluating microencapsulated antibiotics in
animals have demonstrated that targeted local release of
antibiotics directly into infected soft tissue and bone via
sustained release of the drug from poly DL(lactide-co-glycolide)
will greatly enhance antibiotic efficacy for both prophylaxis and
treatment. Antibiotic hypersensitivity was, from the beginning, the
most obvious untoward clinical concern of this novel approach to
antibiotic delivery. What effect would sustained antibiotic release
have on the hypersensitive patient?
Prior to the filing of applicants' parent application Ser. No.
590,308 on Mar. 16, 1984, which disclosed the local application of
encapsulated antibiotics to treat wound infection, it was commonly
known that an inherent property of free antibiotics such as
ampicillin, is that they elicit an immune response in man and
induce the production of antibodies. Thus, interest in the immune
response elicited from the sustained release of immunogens
intensified in order to capture the beneficial aspects of this
event immunogenic event in a manner which would advance the
frontiers of medical science. This led to additional studies with
sustain released antibiotics and led the inventors to postulate
that antigens encapsulated in lactide/glycolide could potentially
provide a more effective method of active immunization than free
antigen alone. In follow on experiments, vaccine antigens were
encapsulated and studies were performed to explore this hypothesis
as illustrated in Phase II, herein (Phase II).
VI. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of microencapsulated ampicillin (MEAA) on
the immune response when mice are treated with free ampicillin,
ampicillin encapsulated within biodegradable-biocompafible
microspheres and placebo poly (Lactide/glycolide) microspheres, by
measuring the specific IgG antibodies to free ampicillin and MEAA
in sera of treated mice by ELISA.
FIG. 2 shows that guinea pigs sensitized with free or
microencapsulated ampicillin developed specific IgG antibodies to
ampicillin as measured by ELISA.
FIG. 3 shows the in vitro release of [.sup.14 C]-ampicillin
anhydrate from sterilized microcapsules/spheres (45 to 106
micrometers in diameter) into 0.1 molar potassium phosphate
receiving fluid (pH 7.4) maintained at 37.degree. C. The
microcapsules consisted of about 10 weight percent ampicillin
anhydrate and about 65 weight percent 53:47 DL-PLG polymer.
FIG. 4 shows the in vitro release of [.sup.14 C]-ampicillin
anhydrate from sterilized microcapsules (10 to 100 micrometers
consisting of about 35 weight percent ampicillin and about 65
weight percent of 53:47 DL-PLG polymer.
FIG. 5 shows the mean daily excretion of [.sup.14 C] from rats
receiving subcutaneous injections of sterilized microencapsulated
and unencapsulated [.sup.14 C]-ampicillin anhydrate.
FIG. 6 illustrates that encapsulated as well as the ampicillin
anhydrate showed a fast release of drug during Day 1. By Day 4, the
amount of ampicillin found in the serum of animals dosed with the
unencapsulated drug was below the level of detection of the assay,
whereas serum levels of ampicillin were dectable in animals
receiving encapsulated ampicillin for up to 11 days.
FIG. 7 shows mean serum levels of ampicillin at 1-hour following
implantation of either microencapsulated ampicillin or
unencapsulated ampicillin into the medullary canal of the rabbit
tibia with experimental osteomyelitis. Serum Cefazolin Levels.
FIG. 8 shows the mean serum concentrations of cefazolin that were
measured at 1 hour and 24 hours following local antibiotic therapy
with either CZ microspheres (Group A) or free CZ powder (Group B)
in the rabbit fracture-fixation model. At 1 hour, the mean serum
cefazolin levels were approximately 32 times higher for the Group B
animals who had received local antibiotic therapy with free CZ
powder (18.7.+-.6.1 ug/ml) as compared to the Group A animals who
were treated with CZ microspheres )0.57.+-.0.27 ug/ml). This
difference in the mean serum cefazolin levels between the two
groups was statistically significant (p=0.0023) by Student's t
test. At 24 hours following local treatment, no cefazolin was
detected in the sera of the rabbits who had received free CZ powder
(Group B), however, low cefazolin concentrations were detected in
the sera of Group A animals who were treated with the CZ
microspheres. It is evident from the data that the free antibiotic
diffuses rapidly from the wound and is absorbed into the systemic
circulation, whereas, the microspheres remain localized and
continue to release low but measurable levels of antibiotic for an
extended time interval.
FIG. 9 shows the size destribution of microspheres wherein the
particle size distibution (%) is (a) By number 1-5 (91) and 6-10
(9) and (b) By weight 1-5 (28) and 6-10 (72).
FIG. 10 shows a scanning electron micrograph of microspheres.
FIGS. 11(a) and (b) show the In vitro immunization of spleen cells
and demonstrates that AF/RI pilus protein remains immunogenic to
rabbit spleen cells immunized in vitro after microencapsulation.
AF/R1 pilus protein has been found to be immunogenic for rabbit
spleen mononuclear cells in vitro producing a primary IgM antibody
response specific to AF/RI. Immunization with antigen encapsulated
in biodegradable, biocompatible microspheres consisting of
lactide/glycolide copolymers has been shown to endow substantially
enhanced immunity over immunization with the free antigen. To
determine if microencapsulated AF/RI maintains the immunogenicity
of the free pilus protein, a primary in vitro immunization assay
was conducted. Rabbit spleen mononuclear cells at a concentration
of 3.times.10.sup.5 cells/well. Triplicate wells of cells were
immunized with free AF/RI in a dose range from 15 to 150 ng/ml or
with equivalent doses of AF/RI contained in microspheres.
Supernatants were harvested on days 7, 9, 12, and 14 of culture and
were assayed for free AF/RI pilus protein specific IgM antibody by
the ELISA. Supernatant control values were subtracted from those of
the immunized cells. Cells immunized with free pilus protein showed
a significant positive IgM response on all four days of harvest,
with the antibody response increasing on day 9, decreasing on day
12, and increasing again on day 14. Cells immunized with
microencapsulated pilus protein showed a comparable positive IgM
antibody response as cells immunized with free pilus protein. In
conclusion, AF/RI maintains immunogenicity to rabbit spleen cells
immunized in vitro after microencapsulation.
FIGS. 12(a) and (b) show in vitro immunization of Peyer's patch
cells. Here the AF/RI pilus protein remains immunogenic to rabbit
Peyer's patch cells immunized in vitro after microencapsulation.
AF/RI pilus protein has been found to be immunogenic for rabbit
Peyer's patch mononuclear cells in vitro producing a primary IgM
antibody response specific to AF/RI. Immunization with antigen
encapsulated in biodegradable, biocompatible microspheres
consisting of lactide/glycolide copolymers has been shown to endow
substantially enhanced immunity over immunization with the free
antigen. To determine if microencapsulated AF/RI maintains the
immunogencity of the free pilus protein, a primary in vitro
immunization assay was conducted. Rabbit Peyer's patch mononuclear
cells at a concentration of 3.times.10.sup.6 cells/ml were cultured
in 96-well, round bottom microculture plates at a final
concentration of 6.times.10.sup.5 cells/well. Triplicate wells of
cells were immunized with free AF/RI in a dose range from 15 to 150
ng/ml or with equivalent dose of AF/RI contained in microspheres.
Supernatants were harvested on days 7, 9, 12, and 14 of culture and
were assayed for free AF/RI pilus protein specific IgM antibody by
the ELISA. Supernatant control values were subtracted from those of
the immunized cells. Cells immunized with free pilus protein showed
a significant positive IgM response on all four days of harvest,
with the highest antibody response on day 12 with the highest
antigen dose. Cells immunized with encapsulated pilus protein
showed a positive response on day 12 with all three antigen doses.
In conclusion, AF/RI pilus protein maintains immunogenicity to
rabbit Peyer's patch cells immunized in vitro after
microencapsulation.
FIG. 13 shows proliferative responses to AF/RI by rabbit Peyer's
patch cells. Naive rabbits were primed twice with 50 micrograms of
either non-encapsulated (rabbits 132 and 133) or microencapsulated
(rabbits 134 and 135) AF/RI pili by endoscopic intraduodenal
inoculation seven days apart. Seven days following the second
priming, Peyer's patch cells were cultured with AF/RI in 96-well
plates for four days followed by a terminal six hour pulse with
[.sup.3 H]thymidine. Data shown is the SI calculated from the mean
cpm of quadruplicate cultures. Responses were significant for all
rabbits: 132 (p=0.013), 133 (p=0.0006), 134 (p=0.0016), and 135
(p=0.0026). Responses were significantly different between the two
groups. Comparison of the best responder in the nonencapsulated
antigen group (rabbit 133) with the lowest responder in the
microencapsulated antigen group (rabbit 134) demonstrated an
enhanced response when the immunizing antigen was microencapsulated
(p=0.0034).
Additionally, FIG. 13 relates to the in vitro lymphocyte
proliferation after sensitization of rabbit lymphoid tissues with
encapsulated or non-encapsulated AF/RI pilus adhesion of E. coli
strain RDEC-1. The AF/RI adherence factor is a plasmid encoded
pilus protein that allows RDEC-1 to attach to rabbit intestinal
brush borders. We investigated the immunopotentiating effect of
encapsulating purified AF/RI into biodegradable non-reactive
microspheres composed of polymerized lactide and glycolide,
materials used in resorbable sutures. The microspheres had a size
range of 5-10 microns, a size selected for Peyer's Patch
localizaiton, and contained 0.62% protein by weight. NZW rabbits
were immunized twice with 50 micrograms of either encapsulated or
non-encapsulated AF/RI by intraduodenal later of non-encapsulated
AF/RI by intraduodenal inoculation seven days apart. Lymphocyte
proliferation in respone to purified AR/RI was conducted in vitro
at seven days and showed that encapsulating the antigen into
microspheres enhanced the cellular immune response in the Peyer's
Patch; however, no significant increase was observed in spleen or
mesenteric lymph node. These data suggest that encapsulation of
AF/RI may potentiate the mucosal cellular immune response.
FIGS. 14a-d show proliferative responses to AF/RI synthetic
peptides by rabbit Peyer's patch cells. Naive rabbits were primed
twice with 50 micrograms of either non-encapsulated (rabbits 132
and 133) or microencapsulated (rabbits 134 and 135) AF/RI pili by
endoscopic intraduodenal inoculation seven days apart. Seven days
following the second priming, Peyer's patch cells from each rabbit
were cultured with AF/R1 40-55 (FIG. 14a), AF/R1 79-94 (FIG. 14b)),
AF/R1 108-123 (FIG. 14c), or AF/R1 40-47/79-86 (FIG. 16d) in
96-well plates for four days followed by a terminal six hour pulse
with [.sup.3 H]thymidine. Data shown is the SI calculated from the
mean cpm of quadruplicate cultures. The responses of rabbits 132
and 133 were not significant to any of the peptides tested. Rabbit
134 had a significant response to (a) AF/R1 40-55 (p=0.0001), (b)
AF/R1 79-94 (p=0.0280), and (d) AF/R1 40-57/79-86 (p=0.025), but
not to (c) AF/R1 108-123. Rabbit 135 had a significant response to
(a) AF/R1 40-55 (p=0.034), (b) AF/R1 79-94 (p=0.040), and (c) AF/R1
108-123 (p<0.0001), but not to (d) AF/R1 40-47/79-86. This
demonstrates enhanced proliferative response to peptide antigens
following mucosal priming with microencapsulated pili. AF/RI pili
promotes RDEC-1 attachment to rabbit intestinal brush borders.
Three 16 amino acid peptides were selected by theoretical criteria
from the AF/RI sequence as probable T or B cell epitopes and were
synthesized: AF/RI 40-55 as a B cell epitope, 79-94 as a T cell
epitope, and 108-123 as a T and B cell epitope. We used these
peptides to investigate a possible immunopotentiating effect of
encapsulating purified Af/RI pili into biodegradable, biocompatible
microspheres composed of polymerized lactide and glycolide at a
size range that promotes localization in the Peyer's Patch (5-10
micrometers). NZW rabbits were primed twice with 50 micrograms
AF/RI by endoscopic intraduadenal inoculation and their Peyer's
Patch cells were cultured in vitro with the AF/RI peptides. In two
rabbits which had received encapsulated AF/RI, lymphocyte
proliferation was observed to AF/RI 40-55 and 79-94 in both rabbits
and to 108-123 in one of two rabbits. No responses to any of the
peptides were observed in rabbits which received non-encapsulated
AF/RI. These data suggest that encapsulation of AF/RI may enhance
the cellular response to peptide antigens.
FIGS. 15a-d show B-cell responses of Peyer's patch cells to AF/R1
and peptides.
FIGS. 16a-d show B-cell responses of Peyer's Patch cells to AF/R1
and peptides.
FIGs. 17a-d show B-cell responses of spleen cells to AF/R1 and
Peptides.
FIGS. 18a-d show B cell responses of spleen cells to AF/R1 and
peptides.
FIGS. 15 through 18, illustrate enhanced lymphocyte antibody
response by mucosal immunization of rabbits with microencapsulated
AF/R1 pilus protein. The AF/RI pilus protein has been found to be
immunogenic for rabbit spleen and Peyer's patch cells in vitro
producing a primary IgM antibody response. The purpose of this
study was to determine if AR/R1 pilus protein immune response is
enhanced by microencapsulation. The AF/R1 was incorporated into
biodegradable, biocompatible microspheres composed of
lactide-glycolide copolymers, had a size range of 5-10 micrometer
and containing 0.62% pilus protein by weight. Initially, NZW
rabbits were immunized twice with 50 micrograms of either
encapsulated or non-encapsulated AF/RI via intraduodenal route
seven days apart. For in vitro challenge, 6.times.10.sup.5 rabbit
lymphocytes, were set in microculture at final volume of 0.2 ml.
Cells were challenged with AR/RI or three different synthetic 16
amino acid peptides representing, either predicted T, B or T and B
cell epitopes in a dose range of 15 to 150 ng/ml for splenic cells
or 0.05 to 5.0 micrograms/ml for Peyer's patch mononuclear cells
(in triplicate). Supernatants were collected on culture days 3, 5,
7, and 9 assayed by ELISA for anti-AF/R1 antibody response as
compared to cell supernatant control. Significant antibody
responses were seen only from spleen and Peyer's patch cells from
rabbits immunized with microencapsulated AF/R1. The antibody
response tended to peak between days 5 and 9 was mainly an IgM
response. The results for the predicted epitopes were similar to
those obtained with purified AF/RI. In conclusion, intestinal
immunization with AF/RI pilus protein contained within microspheres
greatly enhances both the spleen and Peyer's patch B-cell responses
to predicted T & B-cell epitopes.
FIG. 19 shows proliferative responses to AF/R1 40-55 by rabbit MLN
cells. Naive rabbits were primed twice with 50 micrograms of either
nonencapsulated (rabbits 132 and 133) or microencapsulated (rabbits
134 and 135) AF/R1 pili by endoscopic intraduodenal inoculation
seven days apart. Seven days following the second priming, MLN
cells were cultured with AF/R1 40-55 for four days in 24-well
plates. Cultures were transferred into 96-well plates for a
terminal [.sup.3 H]thymidine pulse. Data shown is the SI calculated
from the mean cpm of quadruplicate cultures. Responses of rabbits
132 and 133 were not statistically significant. Responses were
significant for rabbits 134 (p=0.0.0051) and 135 (p=0.0055).
FIG. 20 shows proliferative responses to AF/R1 40-55 by rabbit
spleen cells. Naive rabbits were primed twice with 50 micrograms of
either nonencapsulated (rabbits 132 and 133) or microencapsulated
(rabbits 134 and 135) AF/R1 pili by endoscopic intraduodenal
inoculation seven days apart. Seven days following the second
priming, spleen cells were cultured with AF/R1 40-55 for four days
in 24-well plates. Cultures were transferred into 96 well plates
for a terminal [.sup.3 H]thymidine pulse. Data shown is the SI
calculated from the mean cpm of quadruplicate cultures. Responses
of rabbits 132 and 133 were not statistically significant.
Responses were significant for rabbits 134 (p=0.0.0005) and 135
(p=0.0066).
FIG. 21 shows kcpm levels for rabbit spleen cells and mesenteric
lymph node in response to encapsulated AF/R1.
FIG. 22 shows kcpm levels for rabbit spleen cells and mesenteric
lymph node in response to encapsulated AF/R1.
FIG. 23 shows stimulation index responses of rabbit to immunization
with non-encapsulated AF/R1 and encapsulated AF/R1.
FIG. 24. A. SDS-PAGE of intact CFA/I (lane 1), trypsin treated
CFA/I (lane 2), and S. aureus V8 protease treated CFA/I. Molecular
masses of individual bands were estimated from molecular weight
standards (on left). Multiple lanes of both trypsin and V8 treated
CFA/I were transferred to PVDF membranes where bands corresponding
to the approximate molecular masses of 3500 (trypsin digest, see
arrow lane 2) and 6000 (V8 digest, see arrow lane 3) were excised
and subjected to Edman degradation. 24B. Resulting sequence of
protein fragments from each lane of A (position of sequenced
portion of fragment in the intact protein. Underlined, italisized
residues are amino acids under dispute in literature.
FIG. 25. ELISA assay results testing hyperimmune sera of monkeys
25(A)2Z2 (monkey 3), 25(B) 184(D) (monkey 1) and 25(C) 34 (monkey
2) to CFA/I primary structure immobilized on polyethylene pins.
Monkey sera diluted 1:1000. Peptide number refers first amino acid
in sequence of octapeptide on pin from CFA/I primary structure OD
405 refers to optical density wavelength at which ELISA plates were
reat (405 nm).
FIG. 26 Complete sequence of CFA/I (147 amno acids) with B cell
recognition site (boxed areas) as defined by each individual monkey
response (2Z2, 184D, and 34). Derived from data in FIG. 25.
FIGS. 27-29 Lymphocyte proliferation to synthetic decapeptides of
CFA/I. Each monkey was immunized with three i.m. injections of
CFA/I subunits in adjuvant, and its spleen cells were cultured with
synthetic decapeptides which had been constructed using the Pepscan
technique. The decapeptides represented the entire CFA/I protein.
Concentrations of synthetic peptide used included 6.0, 0.6, and
0.06 micrograms/ml. Values shown represent the maximum
proliferative response produced by any of the three concentrations
of antigen used.+-.the standard deviation. The cpm of the control
peptide for each of the three monkeys was 1,518.+-.50, 931.+-.28,
and 1,553.+-.33 respectively. The cpm of the media control for each
of the three monkeys was 1,319.+-.60, 325.+-.13, and 1,951.+-.245
respectively.
FIGS. 30-32 Lymphocyte proliferation to 6.0, 0.6, and 0.06
micrograms/ml synthetic decapeptides of CFA/I in one monkey. The
monkey (2Z2) as immunized with three i.m. injections of CFA/I
subunits in adjuvant, and its spleen cells were cultured with
synthetic decapeptides which had been constructed using the Pepscan
technique. The decapeptides represented the entire CFA/I protein.
Values shown represent the proliferative response which occurred to
6.0 micrograms/ml (FIG. 30), 0.6 micrograms/ml (FIG. 31), or 0.06
micrograms/ml (FIG. 32) of antigen.+-.the standard deviation. The
cpm of the control peptide was 1,553.+-.33 and the cpm of the media
control was 1,951.+-.245.
FIG. 33 shows that rabbits numbers 21 and 22 received intraduodual
administration of AF/R1 microspheres at doses of AF/R1 of 200
micrograms (ug) on day 0 and 100 ug on day 7, 14, and 21 then
sacrificed on day 31. The spleen, Peyer's patch and ileal lamina
propria cells at 6.times.10.sup.5 in 0.2 ml in quadriplate were
challenged with AF/RI and AF/R1 1-13, 40-55, 79-94, 108-123, and
40-47, 79-85 synthetic peptides at 15, 1.5 and 0.15 ug/ml for 4
days. The supernatants were tested for IL-4 using the IL-4/IL-2
dependent cell line cells CT4R at 50,000/well with 0.1 ml of 6.25%
supernatant for 3 days then pulsed with tritiated thymidine for 4
hrs, cells harvested and the tritiated thymidine incorporation
determined, averaged and expressed with one standard deviation
thousand counts per minute (kcpm).
FIG. 34 shows that RDEC-1 colonization (log CFU/gm) in cecal fluids
was similar in both groups (mean 6.3 vs 7.3; p=0.09).
FIG. 35 shows that rabbits given AF/R1-MS remained well and 4/6
gained weight after challenge, whereas 9/9 unvaccinated rabbits
lost weight after challenge (mean weight change +10 vs -270 grams
p<0.01).
FIG. 36 shows that the mean score of RDEC-1 attachment to the cecal
epithelium was zero in vaccinated, and 2+ in unvaccinated
animals.
FIG. 37. Particle size distribution of CFA/II microsphere vaccine
Lot L74F2 values are percent frequency of number or volume verses
distribution. Particle size (diameter) in microns. 63% by volume
are between 5-10 um and 88% by volume are less then 10 um.
FIG. 38. Scanning electron photomicrograph of CFA/II microsphere
vaccine Lot L7472 standard bar represents 5 um distance.
FIG. 39. Twenty-two hour CFA/II release study of CFA/II microsphere
vaccine Lot L7472. Percent cumulative release of CFA/II from three
sample: A, 33.12 mgm; B, 29.50 mgm, 24.20 mgm at 1, 3, 6, 8, 12 and
22 hour intervals. Average represents the mean.+-.ISD.
FIG. 40. Serum IgG antibody reponse to CFA/II microsphere vaccine
Lot L7472 following 2 25 ug protein IM immunization on day 0 in 2
rabbits. Antibody determines on serial dilution of sera by ELISA
and expressed as mean titer versus day 0, 7 and 14.
FIG. 41. Serum IgG antibody response to CFA/II microsphere vaccine
Lot L7F2 following 2 25 ug protein IM immunizations on day 0 if
rabbit 107 & 109. Antibody determined on serial dilution (in
duplicate) of sera by ELISA and expressed as mean titer versus day
0, 7 and 14.
FIG. 42. Lymphocyte proliferative responses for Peyer's patch cells
of rabbits 65 (FIG. 42(a)), 66 (FIG. 42(b)), 83 (FIG 42(c)), 86
(FIG. 42(d)), and 87 (FIG. 42(e)) immunized intraduodenally with 50
mgm protein of CFA/II microsphere vaccine 4 and 7 days earlier. The
cells are challenged in vitro with CFA/II or BSA at 500, 50 and 5
ug/ml or media in triplicate. The uptake of tritiated thymidine in
Kcp is expressed as mean.+-.ISD. Using the paired student t-test,
the p values of 500 ug/ml dose of CFA/II compared to media control
are: 65,p=0.0002; 66,p=0.0002; 83,p=0.0002; and 86, p=0.0002.
FIG. 43. Lymphocyte proliferative responses from Peyer's patch
cells of rabbits 77 (FIG. 43(a)), 78 (FIG. 43(b)), 80 (FIG. 43(c)),
88 (FIG. 43(d)), and 91 (FIG. 43(e)) immunized introduodenally with
50 mgm protein of CFA/II microspheres vaccine 14 and 7 days
earlier. The cells are challenged in vitro with CFA with CFA/II or
BSA at 500, 50 and 5 ug/ml or media in triplicate the uptake of
triciplate. The uptake of tritiated thymidine in Kcp is expressed
as mean.+-.ISD. Using the paired student t -test, the protein of
500 ug/ml dose of CFA/II compared to media control are: 77,
p=0.0001; 78;=0.0015; 80, p=insignificant; 88, p=0.0093; and 91
p=0.0001.
FIG. 44. ELISPOT assay of spleen cells from rabbits 65 (FIG.
44(a)), 66 (FIG. 44(b)), 83 (FIG. 44(c)), 86 (FIG. 44(d)), and 87
(FIG. 44(e)) immunized intraduodenally with 50 mgm protein of
CFA/II microsphere vaccine 14 and 7 days earlier. These were cells
placed into microculture and tested on day 0, 1, 2, 3, 4 and 5 by
ELISPOT for cells secreting antibodies specific for CFA/II antigen.
The results are expressed as number per 9.times.10.sup.6 spleen
cells versus culture day tested.
FIG. 45. ELISPOT assay of spleen cells from normal control rabbits,
67, 69, 72 and 89. The cells were placed into microculture and
tested on days 0, 1, 2, 3, 4 and 5 by ELISPOT for cells secreting
antibodies specific for CFA/II antigen. The results are expressed
as number per 9.times.10.sup.6 spleen cells versus culture day
tested.
FIG. 46. Curve for determining vaccination dosages for regimen
b.
FIG. 47 Hepatitis B surface antigen release from 50:50 poly
(DL-lactide-co-glycolide).
FIG. 48 shows a comparison of drug release from a conventional
system versus a controlled release system. Peak and valley levels
from conventional administrations are shown, in contrast to the
steady therapeutic levels from the controlled release
administration.
FIG. 49 shows a scanning electron micrograph of PLGA microspheres
prepared by the process described in the invention using 50/50
uncapped polymer of Mw 8-12 k dalton and shows superior sphere
morphology, sphere integrity, and narrow size distribution.
FIG. 49a shows a scanning electron micrograph of PLGA microspheres
prepared by conventional solvent evaporation method using a 50/50
uncapped polymer of Mw 8-12 k dalton.
FIG. 50 shows cumulative Histatin release from PLGA microspheres,
wherein release profiles from several batches are prepared using
50/50, uncapped polymer (of Mw 8-12 k dalton) and wherein the
process parameters are varied to modulate release between 1 and 100
days.
FIG. 51 shows a scanning electron micrograph of solid, smooth
spherical surfaces of PLGA microspheres prepared by the method of
in the invention using 50/50, end-capped polymer (of Mw 30-40 k
dalton).
FIG. 52 shows cumulative Histatin release from PLGA microspheres,
wherein the release profiles are from several batches prepared
using 50/50, uncapped and end-capped polymer of Mw 30-40 k daltons,
and wherein the process parameters are varied to modulate release
between 28 to 60 days.
FIG. 53 shows cumulative Histatin release from PLGA o microspheres,
wherein combined release profiles from several batches have been
prepared using 50/50, uncapped and end-capped polymer of Mw 8-40 k
daltons, while varying the process parameters to modulate release
between 1 and 60 days.
FIG. 54 shows a cumulative percent release of LHRH from PLGA
microspheres prepared using uncapped polymer of Mw 8-12
daltons.
VII. DETAILED DESCRIPTION OF THE INVENTION
This invention relates to the encapsulation of active core
materials, especially those which are medically beneficial to the
mammalian animal kingdom, such as biologically active agent(s),
drug(s), or substance(s) within a biodegradable-biocompatible
polymeric matrix.
More precisely, applicants have discovered a medicinally beneficial
composition and process with the following itemized features:
1. A composition for the burst-free, sustained, programmable
release of active material(s) over a period from 1-100 days, which
comprises: (1) An active material and (2) A carrier which may
contain pharmaceutically-acceptable adjuvant, comprised of a blend
of uncapped and end-capped biodegradable-biocompatible
copolymer.
2. The composition of Item 1 wherein the polymeric substance is
poly(lactide/glycolide).
3. The composition of Item 2, wherein the poly(lactide/glycolide)
is a blend of uncapped and end-capped forms, in ratios ranging from
100/0 to 1/99.
4. The composition of Item 3 wherein the copolymer (lactide to
glycolide L/G) ratio for uncapped and end-capped polymer is 90/10
to 40/60.
5. The composition of Item 4 wherein the copolymer (lactide to
glycolide L/G) ratio for uncapped and end-capped polymer is 48/52
to 52/48.
6. The composition of Item 2 wherein the molecular weight of the
copolymer is between 2,000-60,000 daltons.
7. The composition of Item 3 wherein the active material is
biologically active agent.
8. The composition of Item 7 wherein the agent is selected from the
group consisting essentially of antibacterial agents; peptides;
polypeptides; antibacterial peptides; antimycobacterial agents;
antimycotic agents; antiviral agents; hormonal peptides;
cardiovascular agents; narcotic antagonists; analgesics;
anesthetics; insulins; steroids including HIV therapeutic drugs
(including protease inhibitors) and AZT; estrogens; progestins;
gastrointestinal therapeutic agents; non-steroidal
anti-inflammatory agents; parasympathoimetic agents;
psychotherapeutic agents; tranquilizers; decongestants;
sedative-hypnotics; non-estrogenic and non-progestional steroids;
sympathomimetic agents; vaccines; vitamins; nutrients;
anti-migraine drugs; electrolyte replacements; ergot alkaloids;
anti-inflammary agents; prostaglandins; cytotoxic drugs; antigens;
antibodies; enzymes; growth factors; immunomodulators; pheromones;
prodrugs; psychotropic drugs; nicotine; antiblood clotting drugs;
appetite suppressants/stimulants and combinations thereof;
contraceptive agents include estrogens such as diethyl silbestrol;
17-beta-estradiol; estrone; ethinyl estradiol; mestranol;
progestins such as norethindrone; norgestryl; ethynodiol diacetate;
lynestrenol; medroxyprogesterone acetate; dimethisterone; megestrol
acetate; chlormadinone acetate; norgestimate; norethisterone;
ethisterone; melentate; norgestimate; norethisterone; ethisterone;
melengestrol; norethynodrel; and spermicidal compounds such as
nonyphenoxypolyoxyethylene glycol; benzethonium chloride;
chlorindanol; include gastrointestinal therapeutic agents such as
aluminum hydroxide; calcium carbonate; magnesium carbonate; sodium
carbonate and the like; non-steroidal antifertility agents;
parasympathomimetic agents; psychotherapeutic agents; major
tranquilizers such as chloropromaquine HCL; clozapine;
mesoridazine; metiapine; reserpine; thioridazine; minor
tranquilizers such as chlordiazepoxide; diazepam; meprobamate;
temazepam and the like; rhinological decongestants;
sedative-hypnotics such as codeine; phenobarbital; sodium
pentobarbital; sodium secobarbital; other steroids such as
testosterone and testosterone and testosterone propionate;
sulfonmides; sympathomimetic agents; vaccines; vitamins and
nutrient such as the essential amino acids; essential fats;
anti-HIV agents; including AZT; antimalarials such as
4-aminoquinolines; 8 aminoquinolines; pyrimethamine; anti-migraine
agents such as mazindol; phentermine; anti-Parkinson agents such as
L-dopa; antispasmodics such as atropine; methscopolamine bromide;
antispasmodics and anticholingeric agents such as bile therapy;
digestants; enzymes and the like; antitussives such as
dextromethorphan and noscapine; bronchodilators; cardiovascular
agents such as anti-hypertensive compounds; Rauwolfia alkaloids;
coronary vasodilators; nitroglycerin; organic nitrites;
pentaerythriotetranitrate; electrolyte replacements such as
potassium chloride; ergotalkaloids such as ergotamine with and
without caffein; hydrogenated ergot alkaloids; dihydroergocristine
methanesulfate; dihydroergocornine methanesulfonate;
dihydroergokroyptine methaneusulfate and combinations thereof;
alkaloids such as atropine sulfate; Belladonna; hyoscine
hydrobromide; analgesics; narcotics such as codeine;
dihydrocodienone; meperidine; morphine; non-narcotics such as
salicylates; aspirin; acetaminophen; and d-propoxyphene;
antibiotics such as the cephalosporins including ceflacor and
cefuroxime; chloranphenical; gentamicin; Kanamycin A. Kanamycin B;
the penicillins; ampicillin; amoxicillin; streptomycin A; antimycin
A; chloropamtheniol; metromidazole; oxytetracycline penicillin G;
the tetracyclines; including minocycline; fluoro-quinolones
including ciprofloxacin; ofoxacin; macrolides including
clarithromycin; frythromycin; aminioglycosides including
gentamicin; amikacin; tobramycin and kanamycin; beta-lactams
including ampacillin; polymyxin-B; amphotercin-B; aztrofonam;
chloramphenicol; fusidans; lincosamides; metronidazole;
nitro-furantion; imipenem/cilastin; quinolones; systemic antibodies
including rifampin; polygenes; sulfunamides; trimethoprim;
glycopeptides including vancomycin; teicoplanin and imidazoles;
anti-cancer agents; including anti-kaposi's sarcoma;
anti-convulsants such as mephenytoin; phenobarbital; trimethadione;
anti-emetics such as triethylperazine; antihistamines such as
chlorophinazine; dimenhydrinate; diphenhydramine; perphenazine;
tripelennamine and the like; anti-inflammatory agents such as
hormonal agents; hydrocortisone; prednisolone; prednisone;
non-hormonal agents; allopurinol; for claims water-soluble hormone
drugs; antibiotics; antitumor agents; anti inflammatory agents;
antipyretics; analgesics; antitussives; expectorants; sedatives;
muscle relaxants; antiepileptics; anticulcer agents;
antidepressants; antiallergic drugs; cardiotonics; antiarrhythmic
drugs; vasodilators; antihypertensives; diuretics; anticoagulants;
and antinarcotics; in the molecular wight range of 100-100;000
daltons; indomethacin; phenylbutazone; prostaglandins; cytotoxic
drugs such as thiotepa; chloramucil; cyclophosphamide; melphala;
nitrogen mustard; methotrexate; antigens such as proteins;
glycoproteins; synthetic peptides; carbohydrates; synthetic
polysaccharides; lipids; glycolipids; lipopolysaccharides(LPS);
synthetic lipopolysaccharides and with or without attached
adjuvants such as synthetic muramyl dipeptide derivatives; antigens
of such microorganisms as Neisseria gonorrhea; Mycobacterium
tuberculosis; Picarinii Pnfumonia; Herpes virus (humonis types 1
and 2); Herpes zoster; Candidia albicans; Candida tropicalis;
Trichomonas vaginalis; Haemophilus vaginalis; Group B
streptoccoccus ecoli; Microplasma hominis; Hemophilus ducreyi;
Granuloma inguimale; Lymphopathia venerum; Treponema palidum;
Brucela aborus Brucela meitensis Brucela suis; Brucella canis
Campylobacter fetus; Campylobacer fetus intesinalis; Leptospira
pomona; Listeria monocytogenes; Brucella ovis; Equine herpes virus
1; Equine arteritis virus; IBR-IBP virus; Chlamydia psittaci;
Trichomonas foetus; Taxoplasma gondii; Escherichia coli;
Actinobacillus equili; Salmonella abortus ovis. Salmonella abortus
eui; Pseudomonas aeruginosa; Corynebacterium equi; Corynebacterium
pyogenes; Actinobaccilus seminis; Mycoplasma bovigenitalium;
Aspergilus fumigatus, Absidia ramosa; Trypanosoma equiperdum;
Babesia cabali; Clostridium tetani; antibodies which counteract the
above microorganisms; and enzymes including ribonuclease;
neuramidinase; trypsin; glycogen phosphorylase; sperm lactic
dehydrogenase; sperm hyaluronidase; adenossinetriphosphase;
alkaline phosphatase; alkaline phospha esterase; amino peptides;
typsin chymotrypsin amylase; muramidase; acrosomal proteinase;
diesterase; glutamic acid dehydrogense; succunic and dehydrogenase;
beta-glycophosphatase lipase; ATP-ase alpha-peptate
gamma-glutamyiotranspeptidase; sterold-beta-ol-dehydrogenase;
DPN-di-aprorase; and combinations thereof.
9. The composition of Item 8 wherein the agent is selected from the
group consisting essentially of antibacterial agents; antibacterial
peptides; antimycobacterial agents; antimycotic agents; antiviral
agents; antiparasitic agents; antifungal; hormonal peptides;
cardiovascular agents; narcotic antagonist; analgesics;
anesthetics; vaccines; insulins; HIV therapeutic drugs (protease
inhibitors); estrogens; progestins; gastrointestinal therapeutic
agents; non-steroidal anti-inflammatory agents; parasympathoimetic
agents; psychotherapeutic agents; tranquilizers; decongestants;
sedative-hypnotics; non-estrogenic and non-progestional steroids;
sympathomimetic agents; vaccines; vitamins; nutrients;
anti-malarial compounds; anti-migraine drugs; electrolyte
replacements; ergot alkaloids; analgetics; non-narcotics;
anti-cancer agents; anticonvulsants; anti-emetics; antihistamines;
anti-inflammary agents; prostaglandins; cytotoxic drugs; antigens;
antibodies; enzymes; growth factors; immunomodulators; pheromones;
prodrugs; psychotropic drugs; appetite suppresants/stimulants; and
combinations thereof.
10. The composition of Item 8 wherein the agent is a peptide or
polypeptide.
11. The composition of Item 10 wherein the agent is a poly
peptide.
12. The composition of Item 11 wherein the molecular weight of the
polypeptide is between 1,000-250,000 daltons.
13. The composition of Item 12 wherein the polypeptide is histatin
consisting of 12 amino acids and having a molecular weight of
1563.
14. The composition of Item 1 characterized by the capacity to
completely release histatin in an aqueous physiological environment
within from 1 to 40 days with a 100/0 blend of uncapped and
end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to
52/48, and a molecular weight less than 15,000.
15. The composition of Item 14 wherein the histatin can be
completely released within 18 to 40 days and the molecular weight
of the poly(lactide/glycolide) is within the range of 28,000 to
40,000.
16. The composition of Item 2 characterized by the capacity to
release up to 90% of the histatin in an aqueous physiological
environment from 28-70 days with a 1/99 blend of uncapped and
end-capped poly(lactide/glycolide) having a L/G ratio of 48/52 to
52/48 and a molecular weight range of 10,000-40,000 daltons.
17. The composition of Item 2 characterized by the capacity to
release up to 80% of histatin in an aqueous physiological
environment from 56-100 days with a 1/99 blend of uncapped and
end-capped poly(lactide/glycolide) having a L/G ratio of 75/25 and
a molecular weight of less than 15,000 daltons.
18. The composition of Item 13 having analogs of histatin with
chain lengths of from 11-24 amino acids of molecular weights from
1,500-3,000 daltons and characterized by the following
structures:
1. D S H A K R H H G Y K R K F H E K H H S H R G Y
2. K R H H G Y K R K F H E K H H S H R G Y R
3. K R H H G Y K R K F H E K H H S R
4. R K F H E K H H S H R G Y R
5. A K R H H G Y K R K F H
6. *A K R H H G Y K R K F H
7. K R H H G Y K R K F
*D-amino acid
19. The composition of Item 10 wherein the biologically active
agent is a polypeptide Leutinizing hormone releasing hormone (LHRH)
that is a decapeptide of molecular weight 1182 in its acetate form,
and having the structure:
p- E H W S Y G L R P G
20. The composition of Item 13 having a molecular weight of from
1,000 to 250,000 daltons.
21. The composition of Item 2 wherein release profiles of variable
rates and durations are achieved by blending uncapped and capped
microspheres as a cocktail in variable amounts.
22. The composition of Item 2 wherein release of profiles of
variable rates and duration are achieved by blending uncapped and
capped polymer in different ratios within the same
microspheres.
23. The composition of Item 12 wherein the entrapped polypeptide is
any of the vaccine agents against enterotoxigenic E. coli (ETEC)
selected from the group consisting of CFA/I,CFA/II,CS1,CS3,CS6 and
CS17, ETEC-related enterotoxins, and combinations thereof.
24. The composition of Item 23 wherein the entrapped polypeptide
consists of peptide antigens of molecular weight range of about
800-5000 daltons for immunization against enterotoxigenic E. coli
(ETEC).
25. The composition of Item 24 wherein the entrapped polypeptide is
selected from the group consisting essentially of an antigenic
synthetic peptide containing CFA/I pilus protein T-cell epitopes;
B-cell epitopes, or mixtures thereof.
26. The composition of Item 24 wherein the poly(lactide/glycolide)
is a blend of uncapped and end-capped forms, in ratios ranging from
48/52 to 52/48.
27. The composition of Item 7 wherein said agent are selected from
the group consisting of water-soluble hormone drugs, antibiotics,
antitumor agents, antiinflammatory agents, antipyretics, analgesics
antitussives, expectorants, sedatives, muscle relaxants,
antiepileptics, antiulcer agents, antidepressents, antiallergic
drugs, cardiotonics, antiarrhythmic drugs, vasodilators,
antihypertensives, diuretics, anticoagulants, antinarcotics, in the
molecular weight range of 100-100,000 daltons.
28. The composition of Item 1 wherein said biodegradable
poly(lactide/glycolide) is in an oil phase, and is present in about
1-50% (w/w).
29. The composition of Item 28 wherein concentration of the active
agent is in the range of 0.1 to about 60% (w/w).
30. The composition of Item 29 wherein a ratio of the inner aqueous
to oil phases is about 1/4 to 1/40(v/v).
31. The composition of Item 11 wherein the entrapped polypeptide is
active at a low pH, such as LHRH, adrenocorticotropic hormone,
epidermal growth factor, calcitonin released polypeptide is
bioactive.
32. The composition of Item 11 when entrapped polypeptide such as
histatin is inactive at a low pH, a pH-stabilizing agent of
inorganic salts are added to the inner aqueous phase to maintain
biological activity of the released peptide.
33. The composition of Item 11 wherein when entrapped polypeptide
such as histatin is inactive at a low pH, a non-ionic surfactant
such as polyoxyethylene sorbitan fatty acid esters (Tween 80, Tween
60 and Tween 20) and polyoxyethylene--polyoxypropylene block
copolymers (Pluronics) is added to the inner aqueous phase to
maintain biological activity of the released polypeptide.
34. The composition of Item 32 wherein placebo spheres loaded with
the pH-stabilizing agents are coadministered with
polypeptide-loaded spheres to maintain the solution pH around the
microcapsules and preserve the biological activity of the released
peptide in instances where the addition of pH-stablizing agents in
the inner aqueous phase is undesirable for the successful
encapsulation of the acid pH sensitive polypeptide.
35. The composition of Item 33 wherein placebo spheres loaded with
non-ionic surfactant are coadministered with polypeptide-loaded
spheres to maintain biological activity of the released peptide
where the addition of non-ionic surfactants in the inner aqueous
phase is undesirable for successful encapsulation of the acid pH
sensitive polypeptide.
36. The composition of Item 1 comprising a blend of uncapped and
capped polymer, wherein complete solubilization of the copolymer
leaves no residual polymer at the site of administration and occurs
concurrently with the complete release of the entrapped agent.
37. A process of using composition of Item 1 for human
administration via parenteral routes, such as intramuscular and
subcutaneous.
38. A process of using the composition of Item 1 for human
administration via topical route.
39. A process of using the composition of Item 1 for human
administration via oral routes.
40. A process of using the composition of Item 1 for human
administration via nasal, transdermal, rectal, and vaginal
routes.
41. A process of using the composition of Item 1 for human
administration in the form of an oral or nasal inhalant for the
respiratory tract.
42. A process for preparing controlled release compositions
characterized by burst-free, sustained, programmable release of
biologically active agents, comprising: Dissolving biodegradable
poly(lactide/glycolide), in uncapped form in methylene chloride,
and dissolving a biologically active agent or active core in water;
adding the aqueous layer to the polymer solution and emulsifying to
provide an inner water-in-oil (w/o)emulsion; stabilizing the w/o
emulsion in a solvent-saturated aqueous phase containing a
oil-in-water (o/w) emulsifier; adding said w/o emulsion to an
external aqueous layer containing oil-in-water emulsifier to form a
ternary emulsion; and stirring the resulting water-in-oil-in-water
(w/o/w) emulsion for sufficient time to remove said solvent, and
rinsing hardened microcapsules with water and lyophilizing said
hardened microcapsules.
43. The process of Item 42 wherein a solvent-saturated external
aqueous phase is added to emulsify the inner w/o emulsion prior to
addition of the external aqueous layer, to provide microcapsules of
narrow size distribution range between 0.05-500 um.
44. The process of Item 42 wherein a low temperature of about 0-4
degree C. is provided during preparation of the inner w/o emulsion,
and a low temperature of about 4-20 degree C. is provided during
preparation of the w/o/w emulsion to provide a stable emulsion and
high encapsulation efficiency.
45. A process for preparing controlled release compositions
characterized by burst-free, sustained compositions characterized
by burst-free, sustained, programmable release of biologically
active agents, comprising:
dissolving biodegradble poly(lactide/glycolide) in end-capped form
in methylene chloride, and dissolving a biologically active agent
or active core in water; adding the aqueous layer to the polymer
solution and emulsifying to provide an inner water-in-oil emulsion;
stabilizing the w/o emulsion in a solvent-saturated aqueous phase
containing a oil-in-water (o/w) emulsifier; adding said w/o
emulsion to an external aqueous layer containing oil-in-water
emulsifier to form a ternary emulsion; and stirring a resulting
water-in-oil-water (w/o/w) emulsion for sufficient time to remove
said solvent; and rinsing heardened microcapsules with water; and
lyophilizing said hardened microcapsules.
46. The process of Item 42 wherein a 100/0 blend of uncapped and
end-capped polymer is used to provide release of the active core in
a continuous and sustained manner without a lag phase.
47. The process of Item 45 wherein a solvent-saturated external
aqueous phase is added to emulsify the inner w/o emulsion prior to
addition of the external aqueous layer, to provide microcapsules of
narrow size distribution range between 0.05.500 um.
48. The process of Item 45 wherein a low temperature of about 0-4
degree C. is provided during preparation of the inner w/o emulsion,
and a low temperature of about 4-20 degree C. is provided during
preparation of the w/o/w emulsion to provide a stable emulsion and
high encapsulation efficiency.
49. A method for the protection against infection of a mammal by
pathogenic organisms comprising administering orally to said mammal
an immunogenic amount of an immunostimulating composition
consisting essentially of an antigenic synthetic peptide
encapsulated within a poly(lactide/galactide) matrix.
50. The method of Item 49 wherein the poly(lactide/glycolide) is a
blend of uncapped and end-capped forms, in ratios ranging from
100/0 to 1/99.
51. The method of Item 49 wherein the poly(lactide/glycolide) is a
blend of uncapped and end-capped forms in ratios ranging from 90/10
to 40/60.
52. The method of Item 49 wherein the infection is a bacterial
infection.
53. The method of Item 49 where the synthetic peptide contains an
epitope selected from the group consisting of CFA/I pilus protein
T-cell epitopes, B-cell epitopes or mixtures thereof.
54. The method of Item 49 wherein the infection is a viral
infection.
55. The method of Item 49 wherein the infection is parasitic
infection.
56. The method of Item 49 wherein the infection is a fungal
infection.
57. The method of Item 52 wherein the bacterial infection is caused
by a bacteria selected from the group consisting essentially of
Salmonella typhi, Shigella Sonnei, Shigella Flexneri, Shigella
dysenteriae, Shigella boydii, Escheria coli, Vibrio cholera, Group
D-2, Group E, Group G, Group I, Group 1, Listeria, Erysipelothrix,
Mycobacterium, Aerobic pathogenic Actinomycetales,
Enterobacteriaceae, Vibrio, aeromonas, Plesiomonas, Helicobacter,
W. succinogenes, Acineto bacter spp., Foavobacterium, Pseudomonas,
Legionella, Brucella, Haemophilus, Bordetalla, Mycoplasmas,
Gardnerella, Streptobacillus, Spirillum, Calymmatobacterium,
Clostridium, Treponema, Borrelia, Leptospira, Anaerobic
Gram-negative Bacteria including bacilli and Cocci, Anaerobic
gram-Positive Nonsporeforming Bacilli and Cocci, yersinia,
staphylococcus, clostridium, Enteroccus, Streptoccus, Aerococcus,
Planococcus, Stomatococcus, Micrococcus, Lactoccus, Germella,
Pediococcus, Leuconostoc, Bacillus, Neisseria, Branhamella, Coryne
bacterium, campylobacter, Arcanobacterium haemolyticum, Rhodococcus
spp., Rhodococcus, Group A-4.
58. The method in accordance with Item 49 comprising administering
orally to said mammal an immunogenic amount of a pharmaceutical
composition consisting essentially of an antigenic synthetic
peptide in the amount of 0.1 to 1%.
59. A vaccine for the immunization of a mammal against infection
caused by pathogenic organisms prepared from the composition of
Item 1.
60. The vaccine according to Item 59 wherein the polymeric
substance is poly(DL-lactide-co-glycolide).
61. The vaccine according to Item 60 wherein the relative ratio
between the lactide and glycolide (L/G) component is within the
range of 40/60 to 0/100.
62. The vaccine according to Item 61 wherein the relative ratio
between the amount of lactide and glycolide component is within the
range of 90/10 to 40/60.
63. A vaccine according to Item 62 wherein the pathogenic organisms
are bacterial.
64. A vaccine according to Item 62 wherein the pathogenic organisms
are viral.
65. A vaccine according to Item 62 wherein the pathogenic organisms
are fungal.
66. A vaccine according to Item 62 wherein the pathogenic organisms
are parasitic.
67. The vaccine according to Item 63 wherein the antigenic
synthetic peptide is selected from the group consisting essentially
of Synthetic Peptides Containing CFA/I Pilus Protein T-cell
Epitopes (Starting Sequence # given)
4(Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro),
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu),
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
26(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro),
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser),
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln),
87(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe),
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr), and
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val), and mixtures
thereof;
Synthetic Peptides Containing CFA/I Pilus Protein B-cell (antibody)
Eptiopes (Starting Sequence # given)
3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val),
11(Val-Asp-Pro-Val-Idle-Asp-Leu-Leu-Gln-Ala-Asp),
22(Gly-Asn-Ala-Leu-Pro Ser-Ala-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe),
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser),
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr),
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof; and
Synthetic Peptides Containing CFA/I Pilus Protein T-cell and B-cell
(antibody) Epitopes (Starting Sequence # given)
3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro),
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof.
68. The vaccine according to Item 67 wherein the bacteria is
selected from the group consisting essentially of Salmonella typhi,
Shigella Sonnei, Shigella Flexneri, Shigella dysenteriae, Shigella
boydii, Escheria coli, Vibrio cholera, Group D-2, Group E, Group G,
Group I, Group 1, Listeria, Erysipelothrix, Mycobacterium, Aerobic
pathogenic Actinomycetales, Enterobacteriaceae, Vibrio, aeromonas,
Plesiomonas, Helicobacter, W. succinogenes, Acineto bacter spp.,
Foavobacteriumn, Pseudomonas, Legionella, Brucella, Haemophilus,
Bordetalla, Mycoplasmas, Gardnerella, Streptobacillus, Spirillum,
Calymmatobacterium, Clostridium, Treponema, Borrelia, leptospira,
Anaerobic Gram-negative Bacteria including bacilli and Cocci,
Anaerobic gram-Positive Nonsporeforming Bacilli and Cocci,
yersinia, staphylococcus, clostridium, Enteroccus, Streptoccus,
Aerococcus, Planococcus, Stomatococcus, Micrococcus, Lactoccus,
Germella, Pediococcus, Leuconostoc, Bacillus, Neisseria,
Branhamella, Coryne bacterium, campylobacter, Arcanobacterium
haemolyticum, Rhodococcus, Rhodococcus, Group A-4.
69. The vaccine according to Item 67 wherein the antigenic
synthetic peptide is selected from the group consisting essentially
of 4(Asn-Ile-Thr-Val-thr-Ala-Ser-Val-Asp-Pro),
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu),
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
26(Pro-Ser-ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro),
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser),
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln),
87(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe),
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr), and
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val), and mixtures
thereof.
70. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
4(Asn-Ile-Thr-Val-Thr-Ala-ser-Val-Asp-Pro).
71. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is 8(Thr-ala-ser-Val-AspPro-Val-Ile-asp-Leu).
72. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp).
73. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala).
74. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val).
75. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
26(Pro-Ser-Ala-Val-Lys-Leu-Ala-tyr-Ser-Pro).
76. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser).
77. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln).
78. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
87(Gln-Val-Leu-Ser-Thr-thr-Ala-Lys-Glu-Phe).
79. The vaccine according to claim 69 wherein the antigenic
synthetic peptide is
126(Ala-Gly-Thr-Ala-pro-Thr-Ala-Gly-Asn-Tyr).
80. The vaccine according to Item 69 wherein the antigenic
synthetic peptide is
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val).
81. The vaccine according to Item 67 wherein the antigenic
synthetic peptide is selected from the group consisting essentially
of 3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val),
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
22(Gly-Asn-Ala-Ieu-Pro-Ser-Ala-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe),
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser),
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr),
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
124(Lys-Thr-Ala-Gly-Thr-Ala-ProThr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof.
82. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val).
83. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp).
84. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 22(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val).
85. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val).
86. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe).
87. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val).
88. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser).
89. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala).
90. The vaccine according to Item 81 wherein the antigenic
synthetic peptide is 124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr).
91. The vaccine according to Item 82 wherein the antigenic
synthetic peptide is
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser).
92. The vaccine according to Item 82 wherein the antigenic
synthetic peptide is
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser).
93. The vaccine according to Item 67 wherein the antigenic
synthetic peptide is selected from the group consisting essentially
of 3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro),
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and mixtures
thereof.
94. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro).
95. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-LeuLeu-Gln-Ala-Asp).
96. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-ala-Asp).
97. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val).
98. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser).
99. The vaccine according to Item 93 wherein the antigenic
synthetic peptide is
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser).
100. The method of Item 54, wherein the viral infection is caused
by a virus selected from the group consisting essentially of
hepatitis A, hepatitis B, hepatitis C, Varicella-Zoster virus,
Epstein-Barr virus, Rotaviruses, polio virus, human
immunodeficiency virus (HIV), herpes simplex virus type 1, human
retroviruses, herpes simplex virus type 2, Ebola virus, cytomegalo
viruses, Herpes Simplex viruses, Human cytomegalovirus,
Varicella-Zoster Virus, Epstein-Barr Virus, Poxvirus, Influenza
viruses, Parainfluenza viruses, Respiratory Syncytial virus,
Rhinoviruses, Coronaviruses, Adenoviruses, Measles virus, Mumps
virus, Robella Virus, Human Parvoviruses, Arboviruses, Rabies
virus, Enteroviruses, reoviruses, Viruses Causing gastroenteritis
Hepatitis Viruses, Filoviruses, Arenaaviruses, Papilomaviruses,
Polyomaviruses, Human Immunodeficiency viruses, Human Retroviruses,
and Spongiform Encephalopathies.
101. The method in accordance with Item 49 comprising administering
orally to said mammal an immunogenic amount of a pharmaceutical
composition consisting essentially of an antigen in the amount of
0.1 to 1%.
102. A vaccine for the immunization of a mammal against infection
by pathogenic organisms consisting essentially of an antigen in the
amount of 0.1 to 1% encapsulated within a
biodegradable-biocompatible polymeric poly(DL-lactide-co-glycolide)
matrix wherein the polymer is end-cpped or a blend of uncapped and
end-capped polymers.
103. The vaccine according to Item 100 wherein the polymer is a
blend of end-capped and uncapped polymers.
104. The vaccine according to Item 103 wherein the relative ratio
between the lactide and glycolide component is within the range of
90/10 to 40/60.
105. The vaccine according to Item 103 wherein the relative ratio
between the amount of lactide and glycolide component is within the
range of 48/52 to 52/48.
106. The vaccine according to Item 102 wherein the antigen is a
bacteria or derivatives thereof.
107. The vaccine according to Item 103 wherein the antigen is a
virus or derivatives thereof.
108. The vaccine according to Item 103 wherein the antigens is a
parasite or derivative thereof.
109. The vaccine according to Item 103 wherein the antigen is a
fungus or derivative thereof.
110. The vaccine according to Item 106 wherein the bacteria is
selected from the group consisting essentially of Salmonella typhi,
Shigella Sonnei, Shigella Flexneri, Shigella dysenteriae, Shigella
boydii, Escheria coli, Vibrio cholera, Group D-2, Group E, Group G,
Group I, Group 1, Listeria, Erysipelothrix, Mycobacterium, Aerobic
pathogenic Actinomycetales, Enterobacteriaceae, Vibrio, aeromonas,
Plesiomonas, Helicobacter, W. succinogenes, Acineto bacter spp.,
Foavobacterium, Pseudomonas, Legionella, Brucella, Haemophilus,
Bordetalla, Mycoplasmas, Gardnerella, Streptobacillus, Spirillum,
Calymmatobacterium, Clostridium, Treponema, Borrelia, Leptospira,
Anaerobic Gram-negative Bacteria including bacilli and Cocci,
Anaerobic gram-Positive Nonsporeforming Bacilli and Cocci,
yersinia, staphylococcus, clostridium, Enteroccus, Streptoccus,
Aerococcus, Planococcus, Stomatococcus, Micrococcus, Lactoccus,
Germella, Pediococcus, Leuconostoc, Bacillus, Neisseria,
Branhamella, Coryne bacterium, campylobacter, Arcanobacterium
haemolyticum, Rhodococcus spp., Rhodococcus, Group A4.
111. The vaccine of Item 107 wherein the virus is selected from the
group consisting essentially of hepatitis A, hepatitis B, hepatitis
C, Varicella-Zoster virus, Epstein-Barr virus, Rotaviruses, polio
virus, human immunodeficiency virus (HIV), herpes simplex virus
type 1, human retroviruses, herpes simplex virus type 2, Ebola
virus, cytomegalo viruses, Herpes Simplex viruses, Human
cytomegalovirus, Varicella-Zoster Virus, Epstein-Barr Virus,
Poxvirus, Influenza viruses, Parainfluenza viruses, Respiratory
Syncytial virus, Rhinoviruses, Coronaviruses, Adenoviruses, Measles
virus, Mumps virus, Robefla Virus, Human Parvoviruses, Arboviruses,
Rabies virus, Enteroviruses, reoviruses, Viruses Causing
gastroenteritis Hepatitis Viruses, Filoviruses, Arenaaviruses,
Papillomaviruses, Polyomaviruses, Human Immunodeficiency viruses,
Human Retroviruses, and Spongiform Encephalopathies.
112. An immunostimulating composition comprising
encapsulating-microspheres, which may contain a
pharmaceutically-acceptable adjuvant, wherein said microspheres
having a diameter between 1 nanogram (ng) to 10 microns (um) are
comprised of (a) a biodegradable-biocompatible poly
(DL-lactide-co-glycolide) as the bulk matrix, wherein the copolymer
(lactide to glycolide L/G) ratio for uncapped and end-capped
polymer is 0/100 to 1/99 and (b) an immunogenic substance
comprising a bacteria, virus, fungus, parasite, or derivative
thereof, that serves to elicit the production of antibodies in
animal subjects.
113. An immunostimulating composition according to Item 112 wherein
the amount of said immunogenic substance is within the range of 0.1
to 1.5% based on the volume of said bulk matrix.
114. An immunostimulating composition according to Item 10 wherein
the immunogenic substance comprises Colony Factor Antigen (CFA/II),
hepatitis B surface antigen (HBsAg), a mixture thereof
physiologically similar antigen.
115. An immunostimulating composition according to Item 113 wherein
the relative ratio between the lactide and glycolide component is
within the range of 48/52 to 52/48.
116. An immunostimulating composition according to Item 113 wherein
the size of more than 50% of said microspheres is between 5 to 10
um in diameter by volume.
117. An immunostimulating composition according to Item 113 wherein
the immunogenic substance is the synthetic peptide representing the
peptide fragment beginning with the amino acid residue 63 through
78 of Pilus Protein CS3, said residue having the amino acid
sequence,
63(Ser-Lys-Asn-Gly-Thr-Val-Thr-Try-Ala-His-Glu-Thr-Asn-Asn-Ser-Ala).
118. A vaccine comprising an immunostimulating composition of Item
113 and a sterile, pharmaceutically-acceptable carrier
therefor.
119. A vaccine comprising an immunostimulating composition of Item
118 wherein said immunogenic substance is Colony Factor Antigen
(CFA/II).
120. A vaccine comprising an immunostimulating composition of Item
119 wherein said immunogenic substance is hepatitis B surface
antigen (HBsAg).
121. A method for the vaccination against bacterial infection
comprising administering to a human, an antibactericidally
effective amount of a composition of Item 118.
122. A method according to Item 121 wherein the bacterial infection
is caused by a bacteria selected from the group consisting
essentially of Salmonella typhi, Shigella Sonnei, Shigella
Flexneri, Shigella dysenteriae, Shigella boydii, Escheria coli,
Vibrio cholera, Group D-2, Group E, Group G, Group I, Group 1,
Listeria, Erysipelothrix, Mycobacterium, Aerobic pathogenic
Actinomycetales, Enterobacteriaceae, Vibrio, aeromonas,
Plesiomonas, Helicobacter, W. succinogenes, Acineto bacter spp.,
Foavobacterium, Pseudomonas, Legionella, Brucella, Haemophilus,
Bordetalla, Mycoplasmas, Gardnerella, Streptobacillus, Spirillum,
Calymmatobacterium, Clostridium, Treponema, Borrelia, Leptospira,
Anaerobic Gram-negative Bacteria including bacilli and Cocci,
Anaerobic gram-Positive Nonsporeforming Bacilli and Cocci,
yersinia, staphylococcus, clostridium, Enteroccus, Streptoccus,
Aerococcus, Planococcus, Stomatococcus, Micrococcus, Lactoccus,
Germella, Pediococcus, Leuconostoc, Bacillus, Neisseria,
Branhamella, Coryne bacterium, campylobacter, Arcanobacterium
haemolyticum, Rhodococcus spp. Rhodococcus, Group A-4.
123. A method for the vaccination against viral infection
comprising administering to a human an antivirally effective amount
of a composition of Item 108.
124. A diagnostic assay for bacterial infections comprising a
composition of Item 7.
125. A method of preparing an immunotherapeutic agent against
infections caused by a bacteria comprising the steps of (1)
immunizing a plasma donor with a vaccine according to Item 52 such
that a hyperimmune globulin is produced which contains antibodies
directed against the bacteria; (2) separating the hyperimmune
globulin and (3) purifying the hyperimmune globulin.
126. A method preparing an immunotherapeutic agent against
infections caused by a virus comprising the step of immunizing a
plasma donor with a vaccine according to Item 126 such that
hyperimmune globulin is produced which contains antibodies directed
against the hepatitis B virus.
127. An immunotherapy method comprising the step of administering
to a subject an immunostimulatory amount of hyperimmune globulin
prepared according to Item 125.
128. An immunotherapy method comprising the step of administering
to a subject an immunostimulatory amount of hyperimmune globulin
prepared according to Item 125.
129. A method for the protection against infection of a subject by
enteropathogenic organisms or hepatitis B virus comprising
administering to said subject an immunogenic amount of an
immunostimulating composition of Item 112.
130. A method according to Item 127 wherein the immunostimulating
composition is administered orally.
131. A method according to Item 127 wherein the immunostimulating
composition is administered parenterally.
132. A method according to Item 127 wherein the immunostimulating
composition is administered in four separate doses on day 0, day 7,
day 14, and day 28.
133. A method according to Item 114 wherein the immunogenic
substance is the synthetic peptide representing the peptide
fragment beginning with the amino acid residue 63 through 78 of
Pilus Protein CS3 said residue having the amino acid sequence
63(Ser-Lys-Asn-Gly-Thr-Val-Thr-Try-ala-His-Glu-thr-asn-Asn-Ser-Ala).
134. A method for the protection against or therapeutic treatment
of bacterial infection in the soft tissue or bone of a mammal
comprising administering locally to said mammal a
bactericidally-effective amount of a composition of Item 2, wherein
the active material is an antibiotic which is controlled release
within a period of about 1 to 100 days.
135. The method according to Item 134 wherein the biodegradable
poly(DL-lactide-co-glycolide) is a blend of uncapped and end-capped
forms having a relative ratio between the amount of lactide and
glycolide component within the range of 100/0 to 1/99.
136. A method according to Item 135 wherein the bacterial infection
is (1) a subcutaneous infection secondary to contaminated abdominal
surgery, (2) an infection surrounding prosthetic devices and
vascular grafts, (3) ocular infections, (4) topical skin
infections, (5) orthopedic infections, including osteomyelitis, and
(6) oral infections.
137. The method according to Item 136 wherein the oral infections
are pericoronitis or periodontal disease.
138. The method according to Item 135 wherein the administration is
effected prior to infection.
139. The method according to Item 135 wherein the administration is
effected subsequent to infection.
140. The method according to Item 135 wherein said animal is a
human.
141. The method according to Item 135 wherein said animal is a
nonhuman.
142. The method in accordance with Item 135 comprising applying to
the soft tissue or bone tissue of said animal a
bactericidally-effective amount of a pharmaceutical composition
consisting essentially of an antibiotic in the ant, selected from
the group consisting of a beta-lactam, aminoglycolide, polymyxin-b,
Amphotericin B, Aztreonam, cephalosporins, chloramphenicol,
fusidans, lincosamides, macrolides, methronidazole, nitro-furation,
Imipenem/cilastin, quinolones, refampin, polyenes, tetracycline,
sulfonamides, trimethoprim, vancomycin, teicoplanin, imidazoles,
and erythromycin, encapsulated within a biodegradable
poly(DL-lactide-co-glycolide) polymeric matrix, wherein the amount
of the lactide and glycolide (L/G) component is within the range of
48/52 to 52/48 based on the weight of said polymeric matrix which
is present in the amount of from 40 to 95 percent, resulting in the
controlled release of a bacteriacidal amount of the said antibiotic
over a period of from 1 to 100 days.
143. The method of Item 142 wherein the polymeric matrix consists
essentially of a poly(DL-lactide-co-glycolide) wherein the relative
ratio between the amount of lactide and glycolide (L/G) component
is within the range of 48/52 to 52/48.
144. The method of Item 142 wherein the bacterial infection is
caused by a resistant or non-resistant bacteria selected from the
group consisting essentially of Enterobacteriaceae; Klebsiella sp.;
Bacteroides sp. Enterococci; Proteus sp.; Streptococcus sp.;
Staphylococcus sp.; Pseudomonas sp.; Neisseria sp.;
Pedptostreptococcus sp.; Fusobacterium sp.; Actinomyces sp.;
Mycobacterium sp.; Listeria sp.; Corynebacterium sp.;
Proprionibacterium sp.; Actinobacillus sp.; Aerobacter sp.;
Borrelia sp.; Campylobacter sp.; cytophaga sp.; Pasteurella sp.;
Clostridium sp., Enterobacter aerogenes, Peptococcus sp., Proteus
vulgaris, Proteus morganii, Staphylococcus aureus, Streptococcus
pyogenes, Actinomyces W., Campylobacter fetus, and Legionella
pneumophila, ampillin-resistant strain of S. aureus, and
methicillin-resistant strain of S. aureus.
145. The method of Item 142 wherein the antibiotic is selected from
the group consisting essentially of a beta-lactam, aminoglycolide,
polymyxin-B, amphotericin B, aztreonam, cephalosporins,
chloramphenicol, fusidans, lincosamides, macrolides,
methronidazole, nitro-furantoin, Imipenem/cilastin, quinolones,
rifampin, polyenes, tetracycline, sulfonamides, trimethoprim,
vancomycin, teicoplanin, imidazoles, and erythromycin.
146. The method of Item 145 wherein the beta-lactam is
cephalosporin.
147. The method of Item 145 wherein the beta-lactam is
penicillin.
149. The method of Item 145 wherein the aminoglycolide is
amikacin.
150. The method of Item 145 wherein the aminoglycolide is
tobramycin.
151. The method of Item 145 wherein the aminoglycolide is
kanamycin.
152. The method of Item 145 wherein the beta-lactam is an
ampicillin.
153. The method of Item 152 wherein the polymeric matrix consists
essentially of a poly(DL-lactide-co-glycolide) wherein the relative
ratio between the amount of lactide and glycolide (L/G) component
is within the range of 48/52 to 58/42.
154. The method of Item 152 wherein the ampicillin is present in an
amount of from 5 to 60 percent and the amount of polymeric matrix
is from 40 to 95 percent.
155. The process of using the composition of Item 1 to treat humans
in need, thereof, suffering from diseases and/or ailments from the
group consisting of: viral infections; bacterial infections; fungal
infections; parastic infections and more specific diseases and/or
ailments; such as as, aids; alzheimer's dementia; angiogenesis
diseases; aphthour ulcers in AIDS patients; asthma; atopic
dermatitis; psoriasis; basal cell carcinoma; benign prostatic
hypertrophy; blood substitute; blood substitute in surgery
patients; blood substitute in trauma patients; breast cancer;
breast cancer; cutaneous & metastatic; cachexia in AIDS;
campylobacter infection; cancer; pnemonia; sexually transmitted
diseases (STDs); cancer; viral dieases; candida albicians in AIDS
and cancer; candidiasis in HIV infection; pain in cancer;
pancreatic cancer; parkinson's disease; peritumoral brain edema;
postoperative adhesions (prevent); proliferative diseases; prostate
cancer; ragweed allergy; renal disease; restenosis; rheumatoid
arthritis; rheumatoid arthritis; allergies; rotavirus infection;
scalp psoriasis; septic shock; small-cell lung cancer; solid
tumors; stroke; thrombosis; type I diabetes; type I diabetes
w/kidney transplants; type II diabetes; viseral leishmaniasis;
malaria; periodontal or gum disease; cardiac rthythm disorders;
central nervous system diseases; central nervous system disorders;
cervical dystonia (spasmodic torticollis); choridal
neovascularization; chronic hepatitis c, b and a; colitis
associated with antibiotics; colorectal cancer; coronary artery
thrombosis; cryptosporidiosis in AIDS; cryptosporidium parvum
diarrhea in AIDS; cystic fibrosis; cytomegalovirus disease;
depression; social phobias; panic disorder; diabetic complications;
disabetic eye disease; diarrhea associated with antibiotics;
erectile dysfunction; genital herpes; graft-vs host disease in
transplant patients; growth hormone deficiency; head and neck
cancer; head trauma; stroke; heparin neutralization after cardiac
bypass; hepatocellular carcinoma; HIV; HIV infection; huntington's
disease; CNS diseases; hypercholesterolemia; hypertension;
inflammation; inflammation and angiogensis; inflammation in
cardiopulmonary bypass; influenza; migrain head ache; interstitial
cystitis; kaposi's sarcoma; kaposi's sarcoma in AIDS; lung cancer;
melanoma; molluscum contagiosum in AIDS; multiple sclerosis;
neoplastic meningitis from solid tumors; non-small cell lung
cancer; organ transplant rejection; osteoarthritis; rheumatoid
arthritis; osteoporosis; drug addiction; shock; ovarian cancer;
Amebiasis; Babesiasis; Chagas' disease Trypanosoma cruzi);
Cryptosporidiosis; Cysticercosis; Fascioliasis; Filariasis;
Echinococcosis; Giardiasis; Leishmaniasis; Malaria; Paragonimiasis;
Pneumocystosis; Schistosomiasis; Strongylodiasis; Toxocariasis;
Toxoplasmosis; Trichinellosis; Trichomoniasis; yeast infection; and
pain.
156. A vaccine for prepared from the composition of Item 1 to
prevent the occurence in humans of diseases and/or ailments
comprising viral infections; bacterial infections; fungal
infections; parastic infections and more specific diseases and/or
ailments; such as as, aids; alzheimer's dementia; angiogenesis
diseases; aphthour ulcers in AIDS patients; asthma; atopic
dermatitis; psoriasis; basal cell carcinoma; benign prostatic
hypertrophy; blood substitute; blood substitute in surgery
patients; blood substitute in trauma patients; breast cancer;
breast cancer; cutaneous & metastatic; cachexia in AIDS;
campylobacter infection; cancer; pnemonia; sexually transmitted
diseases (STDs); cancer; viral dieases; candida albicians in AIDS
and cancer; candidiasis in HIV infection; pain in cancer;
pancreatic cancer; parkinson's disease; peritumoral brain edema;
postoperative adhesions (prevent); proliferative diseases; prostate
cancer; ragweed allergy; renal disease; restenosis; rheumatoid
arthritis; rheumatoid arthritis; allergies; rotavirus infection;
scalp psoriasis; septic shock; small-cell lung cancer; solid
tumors; stroke; thrombosis; type I diabetes; type I diabetes
w/kidney transplants; type II diabetes; viseral leishmaniasis;
malaria; periodontal or gum disease; cardiac rthythm disorders;
central nervous system diseases; central nervous system disorders;
cervical dystonia (spasmodic torticollis); choridal
neovascularization; chronic hepatitis c, b and a; colitis
associated with antibiotics; colorectal cancer; coronary artery
thrombosis; cryptosporidiosis in AIDS; cryptosporidium parvum
diarrhea in AIDS; cystic fibrosis; cytomegalovirus disease;
depression; social phobias; panic disorder; diabetic complications;
disabetic eye disease; diarrhea associated with antibiotics;
erectile dysfunction; genital herpes; graft-vs host disease in
transplant patients; growth hormone deficiency; head and neck
cancer; head trauma; stroke; heparin neutralization after cardiac
bypass; hepatocellular carcinoma; HIV; HIV infection; huntington's
disease; CNS diseases; hypercholesterolemia; hypertension;
inflammation; inflammation and angiogensis; inflammation in
cardiopulmonary bypass; influenza; migrain head ache; interstitial
cystitis; kaposi's sarcoma; kaposi's sarcoma in AIDS; lung cancer;
melanoma; molluscum contagiosum in AIDS; multiple sclerosis;
neoplastic meningitis from solid tumors; non-small cell lung
cancer; organ transplant rejection; osteoarthritis; rheumatoid
arthritis; osteioporosis; drug addiction; shock; ovarian cancer;
Amebiasis; Babesiasis; Chagas' disease (Trypanosoma cruzi);
Cryptosporidiosis; Cysticercosis; Fascioliasis; Filariasis;
Echinococcosis; Giardiasis; Leishmaniasis; Malaria; Paragonimiasis;
Pneumocystosis; Schistosomiasis; Strongylodiasis; Toxocariasis;
Toxoplasmosis; Trichinellosis; Trichomoniasis; yeast infection; and
pain.
As noted, in the Summary of the Invention section herein, a
discussion of this invention will be presented as Phases I, II and
III.
Phase I
This illustrative phase of the invention presents the novel use of
a pharmaceutical composition, a micro- or macrocapsule/sphere
formulation, which comprises an antibiotic encapsulated within a
biodegradable polymeric matrix such as poly
(DL-lactide-co-glycolide) (DL-PLG) in the effective pretreatment of
mammals to prevent bacterial infections and the posttreatment of
mammals (including humans and non-human mammals) with bacterial
infections. Microcapsules and microspheres are usually powders
consisting of spherical particles of 2 millimeter or less in
diameter, usually 500 micrometer or less in diameter. If the
particles are less than 1 micron, they are often referred to as
nanocapsules or nanospheres. For the most part, the difference
between microcapsules and nanocapsules is their size; their
internal structure is about the same. Similarly, the difference
between microspheres and nanospheres is their size; their internal
structure is about the same.
A microcapsule (or nanocapsule) has its encapsulated material,
herein after referred to as agent, centrally located within a
unique membrane, usually a polymeric membrane. This membrane may be
termed a wall-forming material, and is usually a polymeric
material. Because of their internal structure, permeable
microcapsules designed for controlled-release applications release
their agent at a constant rate (zero-order rate of release).
Hereinafter, the term microcapsule will include nanocapsules, and
particles in general that comprise a central core surrounded by a
unique outer membrane.
A microsphere has its agent dispersed throughout the particle; that
is, the internal structure is a matrix of the agent and excipient,
usually a polymer excipient. Usually controlled-release
microspheres release their agent at a declining rate (first-order).
But microspheres can be designed to release agents at a near
zero-order rate. Microspheres tend to be more difficult to rupture
as compared to microcapsules because their internal structure is
stronger. Hereinafter, the term microspheres will include
nanospheres, microparticles, nanoparticles, microsponges (porous
microspheres) and particles in general, with an internal structure
comprising a matrix of agent and excipient.
One can use other terms to describe larger microcapsules or
microspheres, that is, particles greater than 500 micrometer to 7
millimeter or larger. These terms are macrocapsules, macrospheres,
macrobeads and beads. Macrocapsules, macrospheres, macrobeads and
beads will be used interchangably herein.
More particularly, the applicants have discovered efficacious
pharmaceutical compositions wherein the relative amounts of
antibiotic to the polymer matrix are within the ranges of 5 to 60
preferred that relative ratio between the lactide and glycolide
component of the poly(DL-lactide-co-glycolide) is within the range
of 40:60 to 100:0, most preferably. Applicants' most preferred
composition consists essentially of 30 to 40(core loading) and 60
to 70 poly(DL-lactide-co-glycolide) (DL-PLG). However, it is
understood that effective core loads for other antibiotics will be
influenced by the nature of the drug, the microbialetiology and
type of infection being prevented and/or treated. From a biological
perspective, the minimal inflammatory response, is biologically
compatible, and degrades under physiologic conditions to products
that are nontoxic and readily metabolized. Similar polymeric
compositions which afford in vitro release kinetics, as discussed
below for DL-PLG, are considered by applicants to be within the
scope of this invention. Applicants have discovered that antibiotic
encapsulated microcapsules/spheres or macrocapsules/spheres (beads)
having a diameter within the range of about 40 microns to about 7
millimeters to be especially useful in the practice of this
invention.
Surprisingly, applicants have discovered an extremely effective
method of treating bacterial infections of soft-tissue or (bone
osteomyelitis) and preventing these type infections with
antibiotics such as beta-lactams, aminoglycosides, polymyxin-B,
amphotericin B, aztreonam, cephalosporins, chloramphenicol,
fusidans, lincosamides, macrolides, metronidazole, nitro-furantion,
Imipenem/cilastin, quinolones, rifampin, polyenes, tetracycline,
sulfonamides, trimethoprim, vancomycin, teicoplanin, imidazoles,
and erythromycin 1) micro- and macroencapsulated or 2) micro- and
macrospheres formulated within a polymeric matrix such as a
poly(DL-lactide-co-glycolide), which has been formulated to release
the antibiotic at a controlled, programmed rate over a desirable
extended period of time. The microcapsules/spheres have been found
to be effective when applied locally, including topically, to open
contaminated wounds thereby facilitating the release of the
antibiotic from multiple sites within the tissue in a manner which
concentrates the antibiotic in the area of need. Similarly, the
encapsulated antibiotics of this invention both in the
microcapsule/sphere and macrocapsule/sphere (bead) form are
effective for the prevention and treatment of orthopedic infections
that include osteomyelitis, contaminated open fractures, and
exchange revision arthroplasty. The macrocapsules/sphere addition
the option to the surgeon of using the subject invention as a
packing material for dead space. The subject invention offers an
optimal treatment for orthopaedic infections because release of the
antibiotic from the micro- or macrocapsule/sphere is completely
controllable over time; antibiotic can be encapsulated into the
sphere; the sphere can be made of any size; and unlike the
methylmethracrylate beads, the subject invention biodegrades over
time to nontoxic products and does not have to be surgically
removed from the treated site. Since virtually any antibiotic can
be encapsulated into the polymer the instant invention can be used
to sustain release all known antibiotics.
Applicants have discovered and/or contemplate that local
application of microencapsulated or macroencapsulated antibiotic
provides immediate, direct, and sustained dosing which targets the
antibiotic to the pre- or post infected soft-tissue or bone site,
and minimizes problems inherent in systemic drug administration. It
appears to applicants that there is a significant reduction of
nonspecific binding of antibiotic to body proteins, while in route
to targeted sites when the antibiotic has been encapsulated in
accordance with this invention. Additionally, antibiotics with
short half-lives can be used more efficiently, undesirable
side-effects can be minimized, and multiple dosing can be
eliminated. These attributes satisfy a long-felt need to improve
the effectiveness and predictability of drug delivery to accomplish
the desired clinical result in patients.
The ability to concentrate the antibiotic within the wound site
ensures an extended period of direct contact between an effective
antibiotic level and the infecting microorganisms. Many drugs have
a therapeutic range below which they are ineffective and above
which they are toxic. Oscillating drug levels, commonly observed
following systemic administration, may cause alternating periods of
ineffectiveness and toxicity. A single dose of desired therapeutic
range. Applicants have discovered that microencapsulated or
macroencapsulated heavy concentrated doses of antibiotics are
effective for the treatment and prevention of infections caused by
antibiotic-resistant bacteria.
Topical application of the antibiotic microcapsule/sphere
formulation to infected wounds allows local application of the
antibiotic in a single dose, whereby an initial burst of antibiotic
for immediate soft- or hard-tissue perfusion, followed by a
prolonged, effective level of antibiotic is achieved in the tissue
at the wound site. Applicants contemplate herein antibiotic
microcapsules/spheres and macrocapsules/spheres consisting of an
antibiotic and DL-PLG and the summarized results of illustrative
experiments that evaluated the prototype microcapsules in vitro and
in vivo.
The subject invention is successful in preventing and treating (1)
soft-tissue infections, (2) osteomyelitis, and (3) infections
surrounding internally fixed fractures. These results were
confirmed using the microcapsule/sphere form of the encapsulated
antibiotics. The microcapsule/sphere and macrocapsule/sphere are
also of value in numerous other applications including soft-tissue
infections that involve, but are not limited to the prevention and
treatment of (1) subcutaneous infections secondary to contaminated
abdominal surgery, (2) infections surrounding prosthetic devices
and vascular grafts, (3) ocular infections, (4) topical skin
infections, and (5) in oral infections such as pericoronitis and
periodontal disease.
The biodegradation rate of the excipient is controllable because it
is related to the mole ratio of the constituent monomers, the
excipient molecular weight and the surface area of the
microcapsules produced. Microcapsules/spheres with diameters of 250
micrometers or less are aerosol spray. The macrocapsules/spheres
are manually placed in the tissue on bone by the surgeon at the
time of surgical debridement. Due to the unique pharmacokinetic
advantages realized with the continuous delivery of antibiotic into
tissue from a controlled-release vehicle, applicants have found
that a small total dose is required to obtain an optimal
therapeutic effect.
VII. EXAMPLES
The herein offered examples provide methods for illustrating,
without any implied limitation, the practice of this invention in
the treatment of bacterial wound infections.
The profile of the representative experiments have been chosen to
illustrate the antibacterial activity of antibiotic-polymeric
matrix composites.
All temperatures not otherwise indicated are in degrees Celcius
(.degree. C.) and parts or percentages are given by weight.
Material and Methods
A. Mcirocapsules/spheres. The ampicillin anhydrate microspheres
used in these studies (Composite Batch D 856-038-1) consisted of
30.7 wt in a copolymer of 52:48 poly (DL-lactideco-glycolide). The
size of the microspheres ranged from 45 to 150 microns and they
were sterilized with 2.0 Mrad of gamma irradiation.
Animals. New Zealand white rabbits (Dutchland Laboratories, Denver,
Pa.), weighing 2.0 to 2.5 kg each, were used. The animals were
housed in individual cages and were fed a standard laboratory diet.
The experiments described herein were conducted in accordance with
the principles set forth in the Guide for the Care and Use of
Laboratory Animals.
Example 1
Osteomyelitis Model. The technique used to produce osteomyelitis
was a modification of the procedure described previously by Norden.
Briefly, New Zealand white rabbits (2.0-2.5 kg, each) were
anesthetized with ketamine hydrochloride and xylazine and access to
the medullary canal was gained by inserting an 18-guage Osgood
needle (Becton Dickinson Corp., Rutherford, N.J.) into the right
proximal tibial metaphysis. Through this needle was injected 0.1 ml
of 5 Pharmaceuticals, Tenafly, N.J.) followed by injection of
approximately 5.times.10.sup.6 CFU of S. aureus ATCC 6538P. The
hole in the bone was sealed with bone wax and each animal received
a single subcutaneous injection of 3-ml TORBUTROL.TM. (A. J. Buck,
Hunt Valley, Md.) for postoperative pain control. Antibiotic
therapy was then initiated either immediately or was delayed for
7-days as described in detail below.
Example 2
Immediate Antibiotic Therapy. The initial experiment was designed
to evaluate the efficacy of local therapy with microencapsulated
ampicillin for the prevention of experimental osteomyelitis. A
total of 31 rabbits were infected in the right proximal tibia with
sodium morrhuate and S. aureus and treatment was initiated
immediately as follows:
Group A (n=6) received three daily subcutaneous injections (75
mg/kg/day) of aqueous sodium ampicillin (Polycillin-N.TM., Bristol
Laboratories, Syracuse, N.Y.) at 8-hour intervals for 14
consecutive days;]
Group B (n=7) received a single intramedullary injection of 100 mg
of microencapsulated ampicillin containing an equivalent of 30.7 mg
of ampicillin anhydrate. The microcapsules/spheres were suspended
in 0.2-ml of 2 injection vehicle) and were then injected into the
medullary canal through the same needle that was used to inject the
sclerosing agent and bacteria;
Group C (n=4) received a single intramedullary injection of 0.12 ml
(30.7 mg) of aqueous sodium ampicillin (representing the
unencapsulated free drug); and
Groups D, E, and F (n=14) served as controls and received either an
intramedullary injection of placebo microcapsules (100 mg) without
antibiotic; injection vehicle (0.2 ml) without antibiotic; or no
treatment.
The animals were observed for a total of 8-weeks during which time
roentgenograms were obtained to evaluate the progression of the
disease. All surviving animals were euthanized intraveneously at
two months postinfection with T-61 euthanasia solution (1 mg/kg/iv)
and the tibiae were harvested for bacteriological analysis as
described below.
Example 3
Delayed Antibiotic Therapy Without Debridement. In the second
experiment, a total of 30 rabbits were injected in the right
proximal tibia with sodium morrhuate and S. aureus and the
infection was allowed to become established for 7-days. On Day 7,
the animals were reanesthetized and an incision was made over the
patellar tendon to expose the tibial tuberosity. A 5-mm drill hole
was made in the tibial tuberosity and a trocar, measuring
approximately 15 centimeters in length, was inserted into the
medullary canal to obtain a marrow specimen for culture. The
animals were then randomly assigned to the following treatment
groups:
Group A (n=8) received three daily subcutaneous injections of
aqueous sodium ampicillin (75 mg/kg/day) at 8-hour intervals for
14-days;
Group B (n=8) received an intramedullary application of 150 mg of
microencapsulated ampicillin containing an equivalent of 45 mg of
ampicillin anhydrate. The microcapsules were initially suspended in
0.2 ml of the injection vehicle and then aspirated into a sterile
trocar. The trocar was then inserted into the medullary canal
through the drill hole in the tibial tuberosity;
Group C (n=8) received an intramedullary application of 0.18 ml (45
mg) of aqueous sodium ampicillin which was also delivered into the
canal with a trocar; and
Group D (n=6) served as controls and received no treatment.
Following the implantation of the antibiotics into the medullary
canal, the hole in the tibial tuberosity was sealed with bone wax
and the incision site was closed with 3-0 Dexon sutures. All of the
surviving animals were euthanized 8 weeks following the initiation
of treatment and the tibiae were harvested for bacteriological
analysis.
Example 4
Delayed Antibiotic Therapy With Debridement. Because standard
treatment of chronic osteomyelitis requires the surgical removal of
devitalized osseous tissue, the objective of this experiment was to
evaluate the efficacy of local antibiotic therapy with
microencapsulated ampicillin anhydrate when used in conjunction
with debridement. A total of 30 rabbits were injected in the right
proximal tibia with sodium morrhuate and S. aureus and the
infection was allowed to establish for 7 days. On Day 7 each animal
underwent a standardized surgical debridement of the infected
tibia. The animals were anesthetized and an incision was made to
expose the medial aspect of the tibia. A Hall drill was used to
decorticate approximately one-third of the bone thereby creating a
channel that extended the length of the bone. The canal was
thoroughly debrided with a curette and then irrigated with 20 ml of
sterile saline. Cultures of the marrow were obtained at this time
for bacteriological analysis. Immediately following completion of
the debridement procedure, the animals were randomly assigned to
the following treatment groups:
Group A (n=10) received 150 mg of microencapsulated ampicillin
containing an equivalent of 45 mg of ampicillin anhydrate. The
microcapsules were suspended in 0.2-ml of injection vehicle and
were then implanted into the debrided canal with a sterile
trocar;
Group B (n=10) received 45 mg of unencapsulated sodium ampicillin
in powder form which was applied uniformly into the debrided canal;
and
Group C (n=5) and Group D (n=5) served as controls and received
either an intramedullary application of placebo microcapsules (150
mg) without antibiotic or (2) an injection vehicle (0.2 ml) without
antibiotic, respectively.
Immediately following the implantation of the materials into the
medullary canal, the incision site was closed with 3-0 Dexon
sutures and each animal received 3-ml of Torbutrol.TM. or 3
consecutive days for postoperative pain. The animals were
euthanized at 8 weeks following the initiation of treatment and the
tibiae were harvested for bacteriological evaluation.
Example 5
Roentgenographic Evaluation. Radiographs of the infected tibiae
were obtained at various time intervals and were evaluated by a
board certified skeletal radiologist (LMM) using a grading system
that was originally developed by Norden et al. Four radiographic
parameters (sequestrum formation, periosteal reaction, bone
destruction, and extent of disease) were evaluated for each animal
and a numerical value was assigned for each variable. The scores
were then totaled to arrive at an overall radiographic severity
score. The highest total score possible with this grading scheme
was +7 and reflected the maximum degree of radiographic
severity.
Example 6
Cultures of Bone. For bacteriological evaluation, the tibiae were
dissected free of adherent soft-tissue and the surface of the bone
was cleaned with alcohol pads. The bone was then weighed and
crushed to small pieces with a sterile mortar and pestle. The
crushed bone was suspended in 5 ml of sterile saline and serial
10-fold dilutions were prepared in 0.1 Each dilution (0.1 ml) was
then streaked onto both sheep blood agar and mannitol salt agar
plates which were incubated aerobically at 37.degree. C. for 24
hours. The recovery of any S. aureus colonies from the bones was
evidence of a persistent osseous infection and was considered as a
treatment failure.
Example 7
Measurement of Serum Ampicillin Levels. In the experiment where
local antibiotic therapy was used in conjunction with debridement,
serum levels of ampicillin were measured for all of the animals
treated with either an intramedullary application of
microencapsulated ampicillin anhydrate (Group A) or unencapsulated
free drug (Group B). Serum was obtained from all animals at 1 hour,
1 day, and 7 days following the implantation of the antibiotics
into the tibiae and serum ampicillin levels were measured using the
agar-well diffusion assay described previously in detail by Bennett
et al. A standard curve was constructed relating the size of the
zones of inhibition obtained with a series of ampicillin standards
tested against Sarcina lutea ATCC 9341 as the reference organism.
Ampicillin concentrations in the test sera were then calculated
from this standard curve.
Results of Examples 1 Through 7
Immediate Antibiotic Therapy. The results of the initial experiment
showing the effect of immediate parenteral versus local ampicillin
therapy for the prevention of experimental osteomyelitis are
presented in Table 2. Radiographic changes were initially detected
in the control animals (Groups D, E, and F) at 2 weeks
postinfection and consisted predominantly of periosteal reaction.
By 7 weeks, however, the majority of the control animals (75 scores
ranging from +5.25 to +7.00 indicating extensive osseous
involvement. Radiographic evidence of osteomyelitis was absent in
animals that received either a 14 day course of parenteral
ampicillin therapy (Group A) or those that received an
intramedullary injection of unencapsulated ampicillin (Group C).
Only a minimal periosteal reaction was noted at day 42 for Group B
animals that received an intramedullary injection of
microencapsulated ampicillin, however, all other radiographic
parameters were found to be within normal limits. Cultures of the
tibiae at 8 weeks following the initiation of treatment showed that
all of the animals treated with either a 14 day course of
parenteral ampicillin therapy or a single intramedullary injection
of microencapsulated ampicillin had sterile bone cultures. Free
unencapsulated ampicillin, injected locally into the bone, was also
effective and sterilized the tibiae of 3 of 4 (75 In contract, all
13 surviving control animals in Groups D, E, and F developed
culture-positive osteomyelitis with S. aureus counts ranging from
1.3.times.10.sup.6 to 2.0.times.10.sup.7 CFU recovered per gram of
bone.
Delayed Antibiotic Therapy Without Debridement. Table 3 shows the
results of the experiment when antibiotic therapy was delayed for 7
days postinfection and was then initiated without debridement. Of
the 8 animals in Group A that received a 14 day course of
parenteral ampicillin therapy, 6 (75 aureus bone cultures. Only 2
of these animals survived the entire length of the experimental
protocol; six animals died within 1-2 weeks of completing their
antibiotic therapy after developing profuse diarrhea. Of the 7
surviving rabbits in Group C that received an intramedullary
application of 45 mg of unencapsulated ampicillin, 5 (71 with a
single intramedullary application of microencapsulated ampicillin
anhydrate (Group B) sterilized the tibiae of 4 of 8 (50 of S.
aureus recovered from the tibiae of the other animals in this group
as compared with the controls (Group D). All of the control animals
developed osteomyelitis with an average of 2.8.times.10.sup.5 CFU
of S. aureus recovered per gram of bone. A Chi square analysis of
the proportion of animals in each treatment group with positive
bone cultures showed no statistically significant differences among
the groups (p=0.23).
Delayed Antibiotic Therapy With Debridement. In this experiment we
evaluated the effect of local antibiotic therapy when used in
conjunction with debridement for the treatment of a 7-day
established osseous infection. Bacteriological cultures of the
tibiae at the time of debridement (before antibiotic therapy was
initiated) yielded S. aureus in 29 of 30 (97 shown in Table 4, all
10 of the animals in Group A that were treated with debridement
plus microencapsulated ampicillin anhydrate had sterile bone
cultures. In contrast, of the 10 animals in Group B that were
treated with debridement plus unencapsulated ampicillin only 3 had
sterile bone cultures whereas 7 developed culture-positive
osteomyelitis. A Chi squire analysis showed a statistically
significant difference (p<0.01) in the proportion of animals
with sterile bone cultures in the microencapsulated ampicillin
treated group as compared with the group that was treated with the
unencapsulated form of the antibiotic. Debridement alone, without
local antibiotic therapy, was not effective for the treatment of
this established osseous infection with all 10 control animals
(Groups C and D) developing culturepositive osteomyelitis.
Serum Ampicillin Levels. In the experiment where local antibiotic
therapy was initiated in conjunction with debridement, serum
concentrations of ampicillin were measured for all animals that
received either an intramedullary application of microencapsulated
ampicillin anhydrate or an equivalent dose of unencapsulated free
ampicillin. The data is presented in FIG. 1. Serum levels of
ampicillin were only detected at 1-hour after the implantation of
the antibiotics into the tibiae. At this time interval, the mean
serum concentration of ampicillin in the Group B animals that
received 45 mg of unencapsulated ampicillin (0.79+0.24
micrograms/ml) was approximately 7-fold higher than the mean serum
ampicillin concentration of the Group A animals that received an
equivalent dose of the microencapsulated form of the antibiotic
(0.11+0.08 micrograms/ml).
Discussion Related to Examples 1 Through 7
Previous attempts to develop a biodegradable antibiotic delivery
system for the local treatment of bone infections have met with
only limited success. Zilch and Lambiris reported on the treatment
of 46 patients with chronic osteomyelitis using a biodegradable
fibrin-cefotaxim compound that was implanted into the bone at the
time of surgical intervention and reported healing in only 67
limitation of this system was the rapid diffusion of the antibiotic
from the fibrin carrier. High concentrations of cefotaxim could
only be maintained locally in the would exudate for up to 72 hours.
In a separate study, Dahners and Funderburk implanted
gentamicin-loaded plaster of paris into the tibiae of rabbits with
established staphylococcal osteomyelitis. Although they observed
clinical and roentgenographic improvements as compared with
nontreated controls, nevertheless, 80 animals treated with the
gentamicin-loaded plaster of paris developed culture-positive
osteomyelitis. Recently Gerhart et al. evaluated
poly(propylenefumarate-co-methylmethacrylate) (PPF-MMA), as a
potential biodegradable carrier for antibiotics. Following the
subcutaneous implantation of gentamicin- or vancomycin-loaded
cylinders of PPF-MMA in rats, high concentrations of each
antibiotic were detected locally in the wound exudate while serum
antibiotic levels remained low. Although the PPF-MMA appears
promising as a potential biodegradable antibiotic carrier, the
efficacy of this system remains to be demonstrated in an
experimental animal model of osteomyelitis.
In the present application we evaluated biodegradable microspheres
of poly(DL-lactide-co-glycolide), containing 30.7 weight percent
ampicillin anhydrate, in an experimental osteomyelitis model of the
rabbit tibia. In the initial experiment where treatment was
initiated immediately following the injection of S. aureus into the
medullary canal, a single intramedullary injection of 100 mg of
microencapsulated ampicillin effectively prevented the
establishment of osteomyelitis in 100 of the animals tested (Table
2). Although a 14 day course of parenteral ampicillin therapy also
prevented osteomyelitis in all animals, the total dose of
antibiotic administered to these animals (1,050 mg) was 34 times
higher than the dose administered to the animals treated locally
with the ampicillin-loaded microcapsules (30.7 mg).
In the second experiment, where antibiotic therapy was delayed for
7 days and was instituted without debridement, a 14 day course of
parenteral ampicillin therapy resulted in a 75 treatment failure
rate (Table 3). Free unencapsulated ampicillin, implanted locally
into the bone, was also ineffective with 71 these animals
developing culture-proven osteomyelitis. A single intramedullary
application of microencapsulated ampicillin, on the other hand,
sterilized the tibiae of 50 significantly reduced the mean number
of S. aureus colonies recovered from the tibiae of the other
animals in this group. It is noteworthy that all animals treated
locally with microencapsulated ampicillin anhydrate survived the
duration of the experimental protocol without developing adverse
side-effects. In contrast, 6 of 8 (75 parenteral ampicillin died
within 1 to 2 weeks of completing their antibiotic therapy. The
cause of death in these animals was most likely antibiotic-induced
diarrhea resulting from colonization of the normal intestinal flora
by Clostridium difficile, a phenomenon that has been previously
noted with rabbits receiving parenteral ampicillin therapy.
In the final experiment, where local antibiotic therapy was delayed
for 7 days and was instituted in conjunction with debridement, a
100 animals treated with debridement plus microencapsulated
ampicillin (Table 4). In contrast, of the 10 animals treated with
debridement plus an equivalent dose of unencapsulated ampicillin
powder, 70 seen in FIG. 5, at 1 hour after implantation of the
antibiotics into the medullary canal, the mean serum concentration
of ampicillin in the animals receiving unencapsulated ampicillin
was approximately 7 times higher (0.79+0.024 micrograms/ml) than in
the group that was treated with microencapsulated ampicillin
anhydrate (0.11+0.08 micrograms/ml). This finding suggests that the
free unencapsulated drug diffuses rapidly from the site of
administration and does not remain localized for a sufficient time
interval to eradicate the infection. The fact that 70 animals
treated with the unencapsulated form of the drug developed
osteomyelitis substantiates this conclusion. The ampicillin-loaded
microcapsules/spheres, on the other hand, remain localized at the
site of administration thereby continuing to release high
concentrations of the antibiotic over time resulting in the
elimination of the infecting organisms.
Applicants' experimental studies have demonstrated that a
controlled-release and biodegradable antibiotic delivery system was
successful for the eradication of a susceptible organism from an
osteomyelitic focus when used in conjunction with adequate
debridement.
Preparation of Ampicillin Anhydrate Microcapsules
Example 8
About 500 g of a 10 wt alcohol) (PVA) was added to a 1-L (liter)
resin kettle and cooled to 5.degree. C. while being stirred at 650
rpm with a 2.5-in. Teflon turbine impeller driven by a motor and a
control unit. A solution consisting of 5 g of 68:32
poly(DL-lactide-co-glycolide) in a mixture of 40 g of
dichloromethane and 20 g of acetone was prepared in a separate
container and stirred magnetically while in an ice bath. In still
another container, 5 g of ampicillin anhydrate was dispersed in 15
g acetone. This mixture was stirred magnetically and then sonicated
to achieve uniform dispersion of single ampicillin anhydrate
crystals. After sonication, the container was placed in an ice
bath, magnetic stirring was continued, and additional acetone was
added to give a total of 30 g of acetone. After complete
dissolution of the copolymer, the ampicillin-acetone dispersion was
added to the copolymer solution. The resulting mixture was stirred
magnetically in an ice bath for about 30 minutes or until
homogeneous, and it was then added to the reaction flask containing
the aqueous PVA solution. The stir rate was reduced from 650 to 500
rpm after the addition was complete. After 15 minutes, the pressure
was reduced to 550 torr to begin slow evaporation of the organic
solvent (dichloromethane and acetone). The pressure was further
reduced to 250 torr. This pressure was maintained for another 18 to
24 hours. The flask was then opened, the suspension was removed,
and the microcapsules were separated from the PVA solution by
centrifugation. The microcapsules were then washed twice with
water, centrifuged, and washed once more with water and recovered
by filtration. The microcapsules were then dried in vacuo and
separated into various size fractions by sieving. A free-flowing
powder of spherical particles was obtained.
Example 9
Dissolve 1.2 g of 50:50 poly(DL-lactide-co-glycolide) in 102 g of
methylene chloride. Ampicillin anhydrate (0.8 g) is next added to
the stirring copolymer solution. This mixture (dispersion of drug
in the copolymer solution) is then placed in a 200-mL resin kettle
equipped with a true bore stirrer having a 1.5-inch Teflon turbine
impeller driven by a motor. While the mixture is stirring at 700 to
800 rpm, 48 mL of 100 centastoke (cSt) silicone oil is pumped into
the resin kettle to cause the poly(DL-lactide-co-glycolide) to
coacervate and coat the dispersed ampicillin anhydrate particles.
After the silicone oil is added to the resin kettle, the contents
of the kettle are poured into heptane to harden the
microcapsules/spheres. After stirring in the heptane for 2 hours,
the microcapsules/spheres are collected on a funnel an dried. A
free-flowing powder of spherical different sized particles is
obtained.
In Vitro Characterization of Microcapsules/spheres
The core loadings of microcapsules/spheres comprising [.sup.14
C]-ampicillin anhydrate and DL-PLG were measured by liquid
scintillation counting. The core loading of microcapsules/spheres
consisting of unlabeled ampicillin anhydrate and some radiolabeled
ampicillin anhydrate and DL-PLG was measured by using a microbial
assay. In the former instance, microcapsules/spheres (about 15 mg)
were solubilized in 1 mL of 0.5 N dimethyl dialkyl quarternary
ammonium hydroxide in toluene (Soluene-350) at 55.degree. C. for 2
to 4 hours. Then, 14 ml of scintillation cocktail
(1,4-bis[2-(5-phenyloxazolyl]benzene (PPO/POPOP) in toluene) was
added, and the radioactivity was measured with a liquid
scintillation spectrometer. In the latter instance,
microcapsules/spheres (about 15 mg) were placed in 5 mL of
methylene chloride. Following dissolution of the DL-PLG excipient,
the insoluble ampicillin anhydrate was extracted from the methylene
chloride with four volumes of sterile 0.1 M potassium phosphate
buffer (pH 8.0). These aqueous extracts were then assayed for the
antibiotic using Sarcina lutea ATCC 9341 (American Tye Culture
Collection, Rockville, Md.) and the agar-diffusion microbial assay
previously described in the literature by Kavanagh, F. (ed.)
Antibiotic Substances in Analytical Microbiology, Vol. II,
1972.
The in vitro release kinetics of [.sup.14 C]-ampicillin anhydrate
microcapsules/spheres was determined following the placement of 30
mg of microcapsules in an 8-ounce bottle. The release study was
initiated by the addition of 50 mL of receiving fluid consisting of
0.1 m potassium phosphate buffer (pH 7.4). The bottle was then
sealed and placed in an oscillating (125 cycles/minutes) shaker
bath maintained at 37.degree. C. Periodically, a 3-ml aliquot of
the receiving fluid was removed for assay and replaced with a fresh
3-ml aliquot of receiving fluid to maintain a constant volume of
receiving fluid throughout the study. The 3-ml aliquots were
assayed for drug by liquid scintillation counting using 12 ml
Scinti Verse-I (Fisher Scientific Co., Pittsburgh, Pa.). The
cumulative amount of the drug released into the receiving fluid was
calculated.
The in vitro release kinetics of unlabeled ampicillin anhydrate
microcapsules/spheres was determined in the following manner:
A known amount of ampicillin anhydrate microcapsules/spheres (about
4 mg of microencapsulated ampicillin anhydrate) and 5.0 ml of
sterile receiving fluid (0.1 M potassium phosphate buffer, pH 7.4)
were added into dialysis tubing. The ends of the tubing were sealed
with plastic clamps. The clamped dialysis tubing containing the
microcapsules/spheres were placed into a sterile 8-ounce bottle
containing 100 ml of sterile receiving fluid (0.1 M potassium
phosphate buffer, pH 7.4). The bottle was placed in a shaker bath
maintained at 37.degree. C. and shaked at 120 cycles per second
with about 3-cm stroke. The receiving fluid was previously
sterilized in an autoclave for 20 minutes at 121.degree. C. Several
dialysis tubing assemblies were prepared for one release study. At
Days 1, 2, 4, 7, 10, 13, 15, 18, and 25, one assembly was removed
from its receiving fluid and air dried.
After drying the assembly, all particles remaining inside the
dialysis tubing were quantitatively transferred to a sterile, glass
test tube (16 by 125 mm), 5 ml of methylene chloride were added to
dissolve the microcapsules, and the drug was extracted with three
5-ml portions of sterile 0.1 M potassium phosphate buffer (pH 8.1).
The extraction and preparation of the sample (along with controls)
was performed using the procedures for core-loading analysis as
discussed above in the extracted samples and controls using the
microbiological assay. Knowing the amount of microencapsulated drug
initially placed in the dialysis tubing and the amount of drug
remaining in the dialysis tubing after incubation with receiving
fluid, the amount of drug released was determined by calculating
the difference between them.
In Vivo Release Profiles of Ampicillin from
Microcapsules/spheres
The rate and duration of release of ampicillin anhydrate from the
microcapsules/spheres were determined in vivo in rats. In one
experiment, about 50- to 80-mg doses of microencapsulated and
unencapsulated ampicillin anhydrate were sterilized in disposable
syringes with a 2.0- or 2.5-Mrad dose of gamma radiation at dry-ice
temperature. The sterile microcapsules/spheres and unencapsulated
[.sup.14 C]-ampicillin anhydrate were then suspended in about 2.0
mL of an injection vehicle comprising 2 wt percent of commercially
available carboxymethyl cellulose (Type 7LF, Hercules Inc.,
Wilmington, Del.) and 1 wt percent Tween 20 (ICI Americas Inc.,
Wilmington, Del.) in sterile water and autoclaved at 121.degree. C.
for 15 minutes. The microcapsules/spheres were administered
subcutaneously into the mid-back region of lightly anesthestized
(ether), male Sprague-Dawley rats. The rats were fed standard
laboratory food and water ad libidum and were housed in individual
stainless steel cages fitted with metabolism funnels and screens
that separated and collected the feces and urine. The urine from
each rat was collected, weighed, and analyzed for [.sup.14
C]-content by liquid scintillation counting. The actual doses of
microcapsules/spheres or unencapsulated drug administered to each
rat was determined after injection by measuring the amount of drug
residue in each syringe by liquid scintillation counting. The
amount of radioactivity excreted daily by each rat was normalized
by the dose of microencapsulated or unencapsulated ampicillin
anhydrate that each rat actually received. This result was then
plotted as a function of time.
In a second experiment, unlabelled ampicillin anhydrate
microcapsules/spheres were tested in rats. The rats were
administered the microcapsules/spheres in the same manner as that
described in the first experiment. The microbiological assay
described above was used to determine the amount of ampicillin in
the serum of these rats.
In Vivo Efficacy Evaluation of Microcapsules/spheres
Experiments to evaluate the efficacy of prototype
microcapsules/spheres in vivo were performed in 250- to 300-g male,
Walter Reed strain, albino rats that were anesthetized with sodium
pentobarbital. The right hind leg was razor-shaved, scrubbed with
Betadine (The Purdue Frederick Co., Norwalk, Conn.), and swabbed
with 70 length and 1 cm deep was made in the thigh muscle and
contaminated with 0.2 g of sterile dirt. The muscles were
traumatized by uniformly pinching them with tissue forceps, and
then the wounds were inoculated with known quantities of
Staphylococcus aureus ATCC 6538P and Streptococcus pyogenes ATCC
19615. All rats were inoculated on the same day of the experiment
with the same quantitated bacterial suspension to insure uniform
inoculum in all wounds. The artificially contaminated wounds were
treated within 1 hour by layering sterile, pre-weighed amounts of
microencapsulated antibiotic directly on the wounds. Control groups
consisted of animals with wounds that either received no therapy,
were overlaid with placebo (unloaded) microcapsules/spheres, or
were treated with locally applied, powdered unencapsulated
ampicillin anhydrate. Following treatment, all wounds were sutured
closed with 3-0 black silk.
Three groups of 20 rats each were used in an efficacy experiment to
evaluate Microcapsules/spheres A382-140-1 formulated from 70:30
DL-PLG. In this experiment, a group of animals with wounds overlaid
with 0.5 g of unloaded microcapsules/spheres was substituted for
the untreated (no therapy) group evaluated in each succeeding
dose-response experiment. In addition, a group of 20 rats treated
with 0.5 g of ampicillin anhydrate microcapsules/spheres per wound,
and a group of 20 rats treated with 120 mg of locally applied
uncapsulated ampicillin anhydrate powder per wound were evaluated.
Five animals from each group were sacrificed at 2, 6, 8, and 14
days and evaluated for the presence of ampicillin in the serum and
tissue and for the presence of infection.
Two dose-response experiments were performed in which
Microcapsules/spheres A681-31-1, formulated from 70:30 DL-PLG, and
Microcapsules/spheres B213-66-1S, formulated from 53:47 DL-PLG were
evaluated. Seven groups of 15 rats each were treated with the doses
of microcapsules shown in Table I. Each experiment included an
additional group of 15 rats which remained untreated.
In dose-response Experiment I, five animals from each group were
sacrificed at 2, 7, and 14 days and evaluated for ampicillin levels
and number of bacteria present per gram of tissue at each wound
site. Serum ampicillin levels were assayed at 2, 4, 7, and 14 days.
In dose-response Experiment II, five animals from each group were
sacrificed at 7, 14, and 21 days and evaluated for ampicillin
levels and number of bacteria present per gram of tissue. Serum
ampicillin levels were determined at 2, 7, 14, and 21 days.
Microcapsules/spheres in a 45 to 106 micron size range made by the
phase-separation process were evaluated in these experiments. The
ampicillin anhydrate content of the microcapsules/spheres (core
loading), batch number, and ampicillin anhydrate equivalent for
each dose of microcapsules/spheres are shown in Table 1.
In all experiments, bacterial counts were performed on homogenized,
preweighed tissue that had been aseptically removed from the wound
sites. Serial dilutions of the homogenized tissue specimens were
plated on sheep blood agar. Colonies of Staphylococcus aureus could
be easily differentiated from Streptococcus pyogenes on the basis
of colonial morphology. Tissue from varying distances around the
wound site and serum removed by cardiac puncture were assayed for
antibiotic content. This was accomplished by placing discs
saturated with known quantities of serum or tissue homogenates on
the surface of Mueller-Hinton agar which had been previously seeded
with standardized amounts of Sarcina lutea ATCC 9341. Following
incubation at 37.degree. C., inhibition zones were measured.
Freshly diluted stock solutions containing known quantities of
ampicillin anhydrate served as standards. Diameters of the
inhibition zones were converted to antibiotic concentrations using
standard curves generated by plotting the logarithm of the drug
concentration against the zone diameters.
Test Results
Microcapsule/spheres In Vitro Evaluation
Ampicillin anhydrate was microencapsulated with DL-PLG excipient.
DL-PLG is a biocompatible aliphatic polyester that undergoes
random, nonenzymatic, hydrolytic scission of the ester linkages
under physiological conditions to form lactic acid and glycolic
acid. These hydrolysis products are readily metabolized. The
purpose of the DL-PLG is to control the release of the ampicillin
anhydrate from the antibiotic microcapsule/spheres formulation and
to protect the reservoir of ampicillin anhydrate from degradation
before it is released from the microcapsules/spheres. Two DL-PLG
excipients were used in this study. One DL-PLG had a
lactide-to-glycolide mole ratio of 70:30 and the other, 53:47. The
53:47 DL-PLG biodegrades faster than the 70:30 DL-PLG because of
its higher glycolide content.
A phase-separation microencapsulation process afforded
microcapsules/spheres in yields of better than 95 The
microencapsulated ampicillin anhydrated product was a fine,
free-flowing powder. The microcapsules/spheres are relatively
spherical in shape, but have puckered regions. Although these
puckered regions exist, the polymer coating was continuous, and
there was no evidence of any fractures or pinholes on the surfaces
of the microcapsules. Moreover, the photomicrograph obtained by
scanning electron microscopy of ampicillin anhydrate microcapsules
did not show any evidence of free unencapsulated ampicillin
anhydrate crystals either among the microcapsules or protruding
through the surface of the microcapsules.
The drug content (core loading) of the ampicillin anhydrate
microcapsule/sphere formations was measured to assess how much
ampicillin anhydrate was incorporated in the microcapsules and to
determine the bioactivity of the ampicillin anhydrate after it had
been microencapsulated.
In general, the core loading of the 45-to 106 microns size fraction
was similar to the theoretical core loading. The core loading of a
few batches of [.sup.14 C]-ampicillin anhydrate
microcapsules/spheres was determined by microbial assay as well as
by radioassay. Within experimental error, both assays gave similar
results. This indicates that the ampicillin anhydrate was not
inactivated during the microencapsulation process. Also, the core
loading of ampicillin anhydrate microcapsules/spheres was
determined by the microbial assay to determine the effect of 2.5
Mrad of gamma radiation on the microencapsulated drug. The
radiation did not inactivate the drug because the core loading
remained the same. For instance, 19.3 spheres with 70:30 DL-PLG
assayed as 19.0 irradiation and 11.0 DL-PLG assayed as 11.4
irradiated unencapsulated and microencapsulated drug were also
checked by thin layer chromatography. Irradiated and nonirradiated
samples chromatographed the same, again indicating that no
degradation of the drug was caused by gamma radiation at a dose of
2.5 Mrad.
In vitro release measurements were used to identify an ampicillin
anhydrate microcapsule/sphere formulation that would release all of
its drug at a controlled rate over a period of two weeks. The
formulation that displayed the desired in vitro release kinetics
were microcapsules/spheres with diameters of 45 to 106 microns
consisting of about 10 wt percent ampicillin anhydrate (Bristol
Laboratories, Syracuse, N.Y.) and microcapsules/spheres with
diameters of 10 to 100 microns consisting of about 35 wt percent
ampicillin anhydrate (Wyeth Laboratories, West Chester, Pa.) and
about 65 wt percent 53:47 DL-PLG. FIGS. 3 and 4 show the in vitro
release profiles of two samples of these prototype microcapsules.
The microcapsules released a desirable initial burst of drug,
representing about 30. The remaining drug was then released at a
slower controlled rate.
The in vitro release profile of sterilized (2.5 Mrad), 17.6
compared with the release profiles of sterilized (2.0 Mrad), 9.6
and 7.8 DL-PLG (FIG. 3).
Microcapsule/sphere In Vivo Evaluation
Pharmacokinetic studies were performed with unencapsulated
ampicillin anhydrate and the same samples of microcapsules that
were tested in vitro, as previously described. As shown in FIGS. 3
and 4, the unencapsulated drug as well as the microcapsules/spheres
showed a fast release of drug during Day 1. By Day 4, the amount of
ampicillin found in the urine or serum of animals dosed with the
unencapsulated drug was below the level of detection of the assay.
On the other hand, the microcapsule/sphere formulations maintained
an elevated level of drug in the urine or serum for extended
periods. Both samples of microcapsules/spheres made with the 53:47
DL-PLG had similar release profiles and released drug for about two
weeks. As illustrated in FIG. 5, the microcapsules/spheres prepared
with 70:30 DL-PLG released drug for at least four weeks. The
results of these pharmacokinetic studies corroborate results of the
in vivo release studies described. The 53:47 microcapsules/spheres
closely meet the desired target duration of release of two
weeks.
The slow rate of ampicillin release from the 70:30
microcapsules/spheres, as shown in FIG. 5, may be undesirable
because a low level of ampicillin anhydrate released over a long
period may provide favorable conditions for the development of
drug-resistant bacterial strains. This slower release of drug could
be attributed to the slower biodegradation rate of the 70:30
DL-PLG, where the water-soluble ampicillin anhydrate remained
trapped inside the hydrophobic DL-PLG excipient until the excipient
biodegraded completely. More specifically, for
microcapsules/spheres prepared with either the 70:30 or 53:47
DL-PLG, one could speculate that the release of drug is due to
diffusion of the drug through water-filled pores, pores that
enlarge as more and more drug is released and as the DL-PLG
bioerodes.
However, all ampicillin anhydrate microcapsules/spheres formulated
effectively reduced bacterial counts in contaminated wounds. The
most dramatic observation was the rapid elimination of
Streptococcus pyogenes. Streptococcus pyogenes was present in 90
from microcapsule/sphere-treated wounds within 48 hours. All three
of the microcapsule/sphere batches evaluated were equally
successful in eliminating this organism within two days. At 7 days
Staphylococcus aureus remained in all treated wounds; however,
compared to untreated controls, the bacterial count per gram of
tissue decreased by at least 2 log.sub.10 between Days 2 and 7.
This reduction was not observed in untreated controls. In the
efficacy evaluation of microcapsules/spheres A382-140-1, wounds
treated with unloaded DL-PLG microcapsules, as well as those
treated with topical unencapsulated ampicillin anhydrate, remained
infected at 14 days with >10.sup.5 organisms per gram of tissue;
whereas, 60 ampicillin anhydrate were sterile. The wounds of the
remaining 4010.sup.3 organisms per gram of tissue. By 14 days,
regardless of the dose administered (0.5-0.05 g), all wounds
treated with microcapsule/sphere sample A681-31-1 were sterile;
whereas, all untreated wounds remained infected with >10.sup.5
organisms per gram of tissue. At 14 days, all wounds treated with
0.15 g of microcapsules/spheres B213-66-1S were sterile, however,
5.7.times.10.sup.2 Staphylococcus aureus per gram of tissue were
counted in the wounds of one animal treated with a 0.25-g dose of
encapsulated ampicillin anhydrate. This failure was attributed to
an abscess around a suture on the wound surface. All wounds treated
with 0.15 g of microcapsules/spheres (B213-66-1S) were sterile;
however, in the group treated with a 0.05-g dose of
microcapsules/spheres, one wound remained contaminated with
3.6.times.10.sup.4 Staphylococcus aureus per gram of tissue. The
untreated control animals, evaluated in parallel with the
microcapsule/sphere-treated groups, averaged 1.4.times.10.sup.5
Staphylococcus aureus per gram of tissue.
Serum levels of drug were dependent upon the ampicillin anhydrate
reservoir present inside the microcapsules/spheres (core loading),
the dose, and the ampicillin release characteristics.
Administration of 0.25 g of Microcapsules/spheres A681-31-1, which
contained a 45.25 mg ampicillin reservoir per wound, maintained a
serum ampicillin level of 8.0.+-.7.3 microgram/milliliter for up to
4 days post-treatment. A dose twice that amount (90.50 mg
ampicillin equivalent) maintained detectable serum ampicillin for
up to 7 days post-treatment at a serum ampicillin concentration of
15.95.+-.5.0 microgram/milliliter for the first 4 days. Serum
ampicillin was not detected in animals whose wounds were treated
with microcapsule/sphere doses containing an ampicillin equivalent
of 28.50 mg or less. Even though serum ampicillin was not detected
in any animal at 14 days, the tissue levels at this time were above
the minimal inhibitory concentrations required to kill both
infecting organisms in all animals treated with microencapsulated
ampicillin anhydrate. This was true with microcapsule/sphere doses
as low as 0.05 gram per wound. Even though serum ampicillin was not
detected, microbial bioassay for ampicillin in tissue removed from
wounds treated with 0.05 gram of microcapsules/spheres (A681-31-1)
contained a mean (n=5) ampicillin level of 54, 70, and 21
micrograms/gram of tissue at 2, 7, and 14 days, respectively.
Because the minimal inhibitory concentrations of ampicillin
required to kill 95 of Staphylococcus aureus and 97 pyogenes is 0.5
and 0.05 micrograms/milliliter, respectively, it is a reasonable
assumption that a more than adequate therapeutic amount of drug was
present at the wound site throughout the two-week treatment
period.
In vitro release studies performed on microcapsules/spheres
formulated with 70:30 DL-PLG (A382-140-1 and A681-31-1) showed drug
release at an efficacious rate over two weeks, but also at a slower
rate for an additional 50 days. The continued release of low
amounts of antibiotic in wounds after two to three weeks is
undesirable because of the potential to provide favorable
conditions for the emergence of ampicillin resistant organisms in
wounds which might harbor small numbers or bacteria. Therefore, to
reduce or eliminate drug trailing, microcapsules/spheres were
reformulated by encapsulating ampicillin anhydrate within the
faster biodegrading polymer 53:47, DL-PLG (sample B213-66-1S), in
vitro release profiles showed a release of 85 to 92 within two
weeks. On the seventh day following treatment of wounds with 0.15
gram of Microcapsules/spheres B213-66-1S, a mean (n=5) of 162.5 g
of ampicillin per gram of tissue was quantitated. In vitro release
studies suggest that this amount drops rapidly in the second week
so that by 14 days marginal killing concentrations are present. In
vivo analysis of tissue removed from wounds treated 15 days
previously with 0.25 gram of these microcapsules/spheres contained
<1.9 micrograms/gram of ampicillin per gram. Although <0.22
micrograms/gram of ampicillin was detected in wounds treated with
0.15 gram, it was unusual to detect any ampicillin at 14 days in
tissue from wounds treated with 0.05 gram per wound. At 21 days
post-treatment, ampicillin was not detected in any of the
wounds.
No serum levels of ampicillin were detected in any of the rats
treated with Microcapsules/spheres B213-66-1S. This was expected
because lower doses (ampicillin equivalent) were administered.
(Table 1).
B. Cefazolin (CZ) microspheres. The CZ microspheres used in these
studies were produced by Southern Research Institute, Birmingham,
Ala. The microspheres consisted of 77.8 weight % copolymer (50:50
molar ratio of lactide to glycolide) with a core leading dose of
22.2 weight % cefazolin. The size of the microspheres ranged from
90 to 355 um in diameter and they were sterilized with 2.7 Mrad of
gamma radiation. In vitro release kinetic studies showed that
approximately 20% of the cefazolin was released from the
microspheres within 6 hours, with the remainder of antibiotic
release extending over a period of 15 days.
Rat wound infection model. Experimental wounds were surgically
created in the paraspinous muscles of Sprague-Dawley rats following
induction of anesthesia with ketamine and xylazine. Sterile sand
(100 mg) was implanted into the wound site to simulate a foreign
body and the wounds were inoculated with 5.times.10.sup.6 CFU each
of Staphylococcus aureus ATCC 27660 and Escherichia coli ATCC
25922. The minimum inhibitory concentration (MIC) of cefazolin for
each of these organisms was 4 ug/ml and 2 ug/ml, respectively. The
animals were then randomly distributed in 6 groups. Groups A, B,
and C (6 rats per group) received local antibiotic therapy with 50
mg, 250 mg, or 500 mg of CZ microspheres, respectively. The
microspheres were applied directly to the wounds and care was taken
to achieve a relatively uniform distribution of the drug throughout
the wound site. Group D (6 rats) received local antibiotic therapy
with 110 mg of CZ powder. This dose was equivalent to the
core-loading dose of cefazolin contained in 500 mg of CZ
microspheres used to treat the Group C animals. Group E (6 rats)
received systemic antibiotic therapy with cefazolin (30 mg/kg)
which was administered as a single intramuscular bolus immediately
after bacterial contamination of the wounds. Group F (3 rats)
served as controls and received no antibiotic therapy. The wounds
were then closed with surgical staples and the animals were
returned to their cages. On postoperative day # 28, the rats were
euthanized and tissue was obtained from each wound for quantitation
of surviving bacteria. The tissue was weighed, homogenized, and
serial 10-fold dilutions were prepared and plated on blood agar.
The number of bacteria recovered from each wound was quantitated
and expressed as CFFU/g tissue.
Rabbit fracture-fixation model. This study was conducted in two
segments and was designed to evaluate the effect of early as well
as delayed local antibiotic therapy for the prevention of infection
in experimental fractures. In segment I, open fractures were
created in the right tibiae of New Zealand White rabbits after
induction of anesthesia with ketamine and xylazine. The fractures
were then inoculated with 0.5 ml of S. aureus ATCC 27660
(2.0.times.10.sup.7 CFU/ml). Within 30 minutes following bacterial
contamination, the animals were randomly distributed in 5 groups.
Group A (8 rabbits) received local antibiotic therapy with 300 mg
of cefazolin microspheres which was applied directly to the
fracture site and the deep musculature. Group B (8 rabbits)
received local antibiotic therapy with an equivalent dose of CZ
powder. Group C (8 rabbits) received systemic antibiotic therapy
with cefazolin (25 mg/kg/day) for 7 days. Groups D and E (4 rabbits
per group) served as controls and received either local application
of placebo microspheres (without cefazolin) or no treatment,
respectively. The fractures were then reduced and plated with a
4-hole dynamic compression plate. Immediately prior to wound
closure, animals in Groups A and B received an additional dose of
either CZ microspheres (300 mg) or an equivalent dose of CZ powder,
respectively, which was applied directly over the fixation plates
and the periosteal tissue. The wounds were then repaired with
sutures and the animals were returned to their cages. Blood was
obtained within 1 hour and again at 24 hours after treatment from
all Group A and B animals for quantitation of serum cefazolin
levels which was measured by a microbial inhibition bioassay.sup.9.
Eight weeks later, all surviving animals were euthanized and the
tibiae were harvested for bacteriological analysis, the bones were
crushed to small pieces with sterile mortar and pestle and saline
was added to make a particulate suspension. Serial dilutions were
then prepared and streaked on blood agar for bacterial isolation.
The number of S. aureus colonies recovered from each specimen was
quantitated and expressed as CFU/g of bone.
In segment II, fractures were created in the right tibia of 29
rabbits and contaminated with S. aureus as described above. After a
2 hour delay, the animals were randomly distributed in 3 groups.
Group A (10 rabbits) received local antibiotic therapy with 600 mg
of CZ microspheres. Group B (10 rabbits) received local antibiotic
therapy with an equivalent dose of CZ powder. Group C (9 rabbits)
served as controls and received no treatment. The fractures were
then reduced, plated, and the wounds were closed with sutures.
Eight weeks later, the surviving animals were euthanized and the
tibiae were harvested and processed for isolation of bacteria as
described above.
Results
Rat wound infection model. Table 5 shows the effect of local versus
systemic cefazolin therapy on the contamination rate in rat
soft-tissue wounds at 28 days postinfection. Local antibiotic
therapy with CZ microspheres, in doses ranging from 50 to 500 mg
per wound, was highly effective for eliminating both organisms from
the wounds. The maximum effect was achieved in the Group C animals
who received the highest dose of CZ microspheres (500 mg) where E.
coli and S. aureus were eliminated from 100% of the wounds. Even at
the lowest dose used (50 mg/wound), 4 of 6 wounds were rendered
completely sterile. Local antibiotic therapy with free CZ powder
sterilized the wounds in 5 of 6 (83%) animals. In contrast,
systemic administration of cefazolin (30 mg/kg failed to sterilize
the wounds in any of the 6 Group E animals tested.
Rabbit fracture-fixation model. Table 6 shows the results of the
clinical and bacteriological findings at 8 weeks in 25 surviving
rabbits when local or systemic antibiotic therapy with cefazolin
was initiated within 30 minutes following bacterial contamination
of the fractures. Deep infection, defined as the presence of pus on
the fixation plate or in the deep tissues, was noted in 6 of the 7
(86%) control animals in Group D (placebo microspheres) and group E
(no treatment). Cultures of the tibiae from all 7 controls were
positive for S. aureus. of the 5 surviving Group animals who
received a 1 week course of systemic cefazolin therapy, deep
infection was noted in 3 cases and S. aureus was recovered from the
bones of 4 of the 5 animals. In contrast, no clinical evidence of
infection was detected in any of the 7 Group A animals who received
an equivalent local dose of free CZ powder. Cultures of the tibiae
were sterile in 6 of (86%) Group A and 5 of 6 (83%) Group B
animals, respectively. There was a statistically significant
difference in the mean log S. aureus counts of the Group A and
Group B animals and all other groups by analysis of variance
(p<0.05). The mean log S. aureus counts for Group C was also
significantly different from all groups with the exception of Group
E (no treatment).
Table 7 shows the results of the clinical and bacteriological
findings at 8 weeks in 23 surviving rabbits when local antibiotic
therapy was delayed for 2 hours following bacterial contamination
of the fractures. Clinical evidence of infection was present in 5
of 7 (71% control animals in Group C and cultures of the tibiae
yielded S. aureus in all 7 cases. Of the 8 animals in Group B who
received local antibiotic therapy with Cz powder, deep infection
was noted in 4 animals and S. aureus was received in 6 of 8 (75%)
cases. In contrast, none of the 8 animals in Group Aa (CZ
microspheres) developed clinical infections and cultures of the
tibiae were sterile in all cases. One way analysis of variance
showed a statistically significant difference in the mean log S.
aureus counts between Groups A and B (p=0.0014); Groups A and C
(p<0.0001); and Groups B and C (p=0.0269).
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Applicants have developed microencapsulated antibiotics for the
local treatment of contaiminated surgical and traumatic wounds.
Preliminary studies have shown that local application of
biodegradable antibiotic microspheres to experimental wounds that
were contaiminated with resistant bacteria was extremely effective
for prevention of wound infection. This success is attributed to
the significantly higher local tissue antibiotic levels that can be
achieved at the wound site with direct local application of
microencapsulated antibiotics as compared to conventional systemic
antibiotic dosing. The findings of the experimental studies are
summarized below:
1. Ampicillin microspheres effectively prevented infection in 8/11
(73%) animals whose wounds were inoculated with an
ampicillin-resistant strain of s. aureus (MIC=750 ug/ml). Systemic
ampicillin failed in 9/9 (100%) cases.
2. Cefazolin microspheres effectively prevented infection in 5/6
(83%) animals whose wounds were inoculated with a
methicillin-resistant strain of S. aureus which was also resistant
to cefazolin (MIC=64 ug/ml). Systemic cefazolin failed in 5/6 (83%)
cases.
3. It is preferred that a initial release (burst) of the
encapsulated antibiotic occur within the first day and the
remaining antibiotic be released over the next 2 to 3 weeks.
Experimental Design for Rat Soft-Tissue Wound Infection Model
Experimental surgical wounds were created in the paraspinous muscle
of anesthetized Sprague Dawley rats, each weighing between 450 to
550 grams. The wounds were then contaiminated with 100 mg of
sterile sand as an infection-potentiating agent. The wounds were
then inoculated with 5.times.10.sup.6 CFU of S. aureus ATCC 33593.
This is a methicillin-resistant strain of S. aureus which is also
resistant to cefazolin (MIC=64 ug/ml). The animals were then
assigned to the following treatment groups:
Group A (n=6): 500 mg of cefazolin (CZ) microspheres was applied
directly to the wounds. This dose contained 110 mg of cefazolin
equivalent.
Group B (n=6): 110 mg of free CZ powder was applied directly to the
wounds.
Group C (n=6): This group received intramuscular injections of CZ
(30 mg/kg/day) at 8 hour intervals for 7 consecutive days.
Group D (n=3): This group served as controls and did not receive
any antibiotic therapy.
The wounds were then closed with surgical staples and the animals
were returned to their cages for the next 5 weeks. At that time,
the animals were humanely euthanized and tissue was removed from
the wounds and cultured for the presence of bacteria. The
bacteriological data are presented in Table 8.
VIII. Utility
Successful controlled release of bioactive ampicillin anhydrate was
achieved in vitro and in vivo. The prototype microcapsules/spheres
effectively controlled or eliminated Staphylococcus aureus and
Steptococcus pyogenes from infected wounds in rats. Additionally,
the formulation would be effective in the treatment of all
bacterial infections caused by organisms sensitive to the
antibiotic encapsulated including but not limited to
Enterobacteriaceae; Klebsiella sp.; Bacteroides sp.; Enterococci;
Proteus sp.; Streptococcus sp.; Staphylococcus sp.; Pseudomonas
sp.; Neisseria sp.; Pedptostreptococcus sp.; Fusobacterium sp.;
Actinomyces sp.; Mycobacterium sp.; Listeria sp.; Corynebacterium
sp. ; Proprionibacterium sp.; Actinobacillus sp.; Aerobacter sp.;
Borrelia sp.; Campylobacter sp.; Cytophaga sp.; Pasteurella sp.;
Clostridium sp.; Enterobacter aeroqenes; Peptococcus sp.; Proteus
vulgaris; Proteus morganii; Staphylococcus aureus; Streptococcus
polygenes; Actinomyces sp.; Campylobacter fetus; and Legionella
pneumophila. Results indicate that optimal microcapsules/spheres
should exhibit a programmed release of an appropriate concentration
of antibiotic over about a 14 day to about a 6 week time period
after which time the microcapsule/sphere should biodegrade, leaving
no trace of drug or excipient.
Phase II
This illustrative phase of this invention relates to a novel
pharmaceutical composition, a microcapsule/sphere formulation, may
contain a pharmaceutically-acceptable adjuvant that comprises an
antigen encapsulated within a biodegradable polymeric matrix such
as poly (DL-lactide-co-glycolide) (DL-PLG), wherein the relative
ratio between the lactide and glycolide component of the DL-PLG is
within the range of 90:10 to 0:100, and its use, as a vaccine, in
the effective pretreatment of animals (including humans) to prevent
intestinal infections caused by a virus or bacteria. In the
practice of this invention, applicants found that the AF/R1
adherence factor is a plasmid encoded pilus composed of repeating
pilin protein subunits that allows E. coli RDEC-1 to attach to
rabbit intestinal brush borders. To identify an approach that
enhances the immunogenicity of antigens that contact the intestinal
mucosa, applicants investigated the effect of homogeneously
dispersing AF/R1 pili within biodegradable microspheres that
included a size range selected for Peyer's Patch localization. New
Zealand White rabbits were primed twice with 50 micrograms of
either microencapsulated or nonencapsulated AF/R1 by endoscopic
intraduodenal inoculation. Lymphoid tissues were removed and
cellular proliferative responses to AF/R1 and synthetic AF/R1
peptides were measured in vitro. The synthetic peptides represented
possible T and/or B cell epitopes which were selected from the
AF/R1 subunit sequence using theoretical criteria. In rabbits which
had received nonencapsulated AF/R1, Peyer's Patch cells
demonstrated slight but significnt proliferation in vitro in
response to AF/R1 pili but not the AF/R1 synthetic peptides. In
rabbits which had received microencapsulated AF/R1, Peyer's Patch
cells demonstrated a markedly enhanced response to AF/R1 and the
synthetic peptides. Cells from the spleen and mesenteric lymph
nodes responded similarly to AF/R1 pili in both groups of animals,
while there was a greater response to the synthetic peptide AF/R1
40-55 in rabbits that had received microencapsulated AF/R1. These
data demonstrate that microencapsulation of AF/R1 potentiates the
mucosal cellular immune response to both the native protein and its
linear peptide antigens.
A primary mucosal immune response, characterized by antipilus IgA,
follows infection of rabbits with E. coli RDEC-1. However,
induction of an optimal primary mucosal response by enteral
vaccination with pilus antigen depends on immunogenicity of pilus
protein, as well as such factors as its ability to survive
gastrointestinal tract (GI) transit and to target immunoresponsive
tissue. We tested the effect of incorporating AF/R1 pilus antigen
into resorbable microspheres upon its ability to induce primary
mucosal and systemic antibody responses after direct inoculation
into the GI tract. METHODS: rabbits were inoculated with 50
micrograms of AF/R1 pilus antigen alone or incorporated into
uniformaly sized (5-10 microns) resorbably microspheres (MIC) of
poly(DL-lactide-coglycolide). Inoculation was by intra-duodenal
(ID) intubation via endoscopy or directly into the ileum near a
Peyer's patch via the RITARD procedure (with the cecum ligated to
enhance recovery of gut secretions and a reversible ileal tie to
slow antigen clearance). ID rabbits were sacrificed at 2 weeks for
collection of gut washes and serum. RITARD rabbits were bled and
purged weekly for 3 weeks with Co-lyte to obtain gut secretions.
Anti-pilus IgA and IgG were measured by ELISA.
TABLE 9 RITARD- RESULTS: *pos/test RITARD-PILI MIC ID-PILI ID-MIC
Anti-pilus IgA (fluid) *7/8 4/8 1/2 0/3 Anti-pilus IgG (serum) 0/8
3/8 0/2 1/3
Native pilus antigen led to a mucosal IgA resposne in 7/8 RITARD
rabbits. MIC caused a similar response in only 4/8, but the groups
were not statistically different. MIC (but not pili) induced some
systemic IgG responses (highest in animals without mucosal
responses). Results in rabbits inoculated ID were similar for pili,
but no mucosal response to ID-MIC was noted. SUMMARY: Inoculation
with pilus antigen produces a primary mucosal IgA response.
Microencapsulation does not enhance this response, although the
antigen remains immunogenic as shown by measurable mucosal and some
strong serum responses. It must be determined whether priming with
antigen in microspheres can enhance secondary responses.
B Cell Epitope Data
Materials and Methods
CFA/I PURIFICATION--INTACT CFA/I pili were purified from H10407
(078:H-) as described by Hall et al, (1989) [20]. Briefly, bacteria
grown on colonization factor antigen agar were subjected to
shearing, with the shearate subjected to differential
centrifugation and isopycnic banding on cesium chloride in the
presence of N-lauryl sarkosine. CFA/I were dissociated to free
subunits in 6M guanididinium HCl, 0.2 M ammonium bicarbonate (2 hr,
25.degree.), passed through an ultrafiltration membrane (Amicon XM
50 stirred cell, Danvers, Mass.), with concentration and buffer
exchange to PBS on a YM 10 stirred cell (Amicon). Examination of
dissociated pili by electron microscopy demonstrated a lack of
pilus structure.
Protein Sequencing--The primary structure of CFA/I has been
determined by protein sequencing techniques (Klemm, 1982) and
through molecular cloning methods (Karjalainen, et al 1989) [21].
In these two studies there was agreement in all but two of the 147
amino acid residues (at positions 53 and 74). To resolve the
apparent discrepancies, CFA/I was enzymatically digested in order
to obtain internal amino acid sequence. Trypsin or S. aureus V8
protease (sequencing grade, Boehringer Mannheim) was incubated with
CFA/I at a 1:50 w:w ratio (Tris 50 mM, 0.1% SDS, pH 8.5 for 16h at
37.degree. (trypsin) or 24.degree. C. (V8)). Digested material was
loaded onto precast 16% tricine SDS-PAGE gels (Schagger and von
Jagow, 1987) (Novex, Encinitis, Calif.) and run following
manufacturers instructions. Separated samples were
electrophoretically transferred to PVDF membranes (Westrans,
Schleicher and Schuell, Keene, N.H.) following Matsiduria (1987)
using the Novex miniblot apparatus. Blotted proteins were stained
with Rapid Coomassie stain (Diversified Biotech, Newton Centre,
Mass.). To obtain the desired fragment containing the residue of
interest within a region accessible by automated gas phase
sequencing techniques, molecular weights were estimated from
standards of molecular weights 20,400 to 2,512 (trypsin inhibitior,
myoglobin, and myoglobin cyanogen bromide fragments; Diversified
Biotech) using the corrected molecular weights for the myoglobin
fragments as given in Kratzin et al., (1989) [22]. The estimated
molecular weights for the unknown CFA/I fragments were compared to
calculated molecular weights of fragments as predicted for CFA/I
from the sequence of CFA/I as analysed by the PEPTIDESORT program
of a package developed by the University of Wisconsin Genetics
Computer Group. Selected fragments were cut from the PVDF emebrane
and subjected to gas phase sequencing (Applied Biosystem 470,
Foster City, Calif.).
Monkey Immunization--Three rhesus monkeys (Macaca mulatta) were
injected intramuscularly with 250 ug of dissociated CFA/I in
complete Freund's adjuvent and subsequently with two injections of
250 ug of antgen in incomplete Freund's adjuvent at weekly
intervals. Blood was drawn three weeks after primary
immunization.
Peptide Synthesis--Continuous overlapping octapeptides spanning the
entire sequence CFA/I were synthesized onto polyethylene pins by
the method of Geysen et al. [16], also known as the PEPSCAN
procedure. Derivitized pins and software were purchased from
Cambridge Research Biochemicals (Valley Stream, N.Y.). Fmoc-amino
acid pentafluorophenyl esters were purchased from Peninsular
Laboratories (Belmont, Calif.), 1-hydroxybenzotriazole monohydrate
(HYBT) was purchased from Aldrich, and reagent grade solvents from
Fisher. To span the entire sequence of CFA/I with a single amino
acid overlap of from one peptide to the next, 140 total pins were
necessary, with a second complete set of 140 pins synthesized
simultaneously.
ELISA procedure--Sera raised in monkeys to purified dissociated
pili were incubated with the pins in the capture ELISA assay of
Geysen et al., [16] with the preimmune sera of the same animal
tested at the same dilution simultaneously with the duplicate set
of pins. Dilution of sera used on the pins was chosen by initial
titration of sera by standard ELISA assay and immunodot blot assay
against the same antigen.
Results
It was essential to utilize the correct sequence of CFA/I in the
synthesis of the pins for both T- and B-cell experiments to carry
out the studies as planned. At issue were the amino acids at
position 53 and 74; incorrect residues at those positions would
effect 36 of 138 pins (26%) for T-cell epitope analysis and 30 of
140 pins (21%) for B-cell analysis. To resolve the discrepancy in
the literature, purified CFA/I was proteolytically digested
separately with trypsin and with S. aureus V8 protease (V8). These
enzymes were chosen in order to give fragments with the residues of
interest (53 and 74) relatively near to the N-terminus for
automated Edman degradation (preferably 1-15 residues). These
digests were separated on tricine SDS-PAGE gels (FIG. 24A) and
molecular masses of fragments estimated. A fragment of 3459
calculated molecular mass is expected from the trypsin digest
(corresponding to amino acids 62-94) and a fragment of 5889
calculated molecular mass is expected from the V8 digest (residues
42-95). These fragments were located within each digest (arrows in
FIG. 24), and a companion gel with four lanes of each digest was
run, electrophoreticaly transferred to PVDF, the bands excised and
sequenced. N-terminal sequences of each fragment are given in FIG.
24B. The N-terminal eighteen residues from the trypsin fragment
were determined that corresponded to positions 62-79 in CFA/I.
Position 74, a serine residue was consistent with that determined
by Karjalainen et al., (Karjalainen et al., 1989). Nineteen
residues of the V8 fragment were determined, corresponding to
residues 41-60 of the parent protein. The twelfth residue of the
fragment contained an aspartic acid, also consistent with
Karjalainen et al. (1989). All other residues sequenced were
consistent with those published previously (including residues
1-29, not shown). For the following peptide synthesis were
therefore utilized the complete amino acid sequence of CFA/I
consistent with Karjalainen et al., (1989).
Sera from monkeys immunized with CFA/I subunits were tested in a
modified ELISA assay, with the preimmunization sera tested
simultaneously with duplicate pins. Assays results are displayed in
FIG. 25. Monkey 2Z2 (FIG. 2A) responded strongly to six regions of
the CFA/I sequence. Peptide 14 (the octapeptide 14-21) gave the
strongest response with four pins adjacent to it (11, 12, 13, and
15) also appearing to bind significant antibody. The other 2Z2
epitopes are centered at peptides 3, 22, 33, 93, and 124. Monkey
184D (FIG. 17B) also responded strongly to peptide 14, although the
maximum response was to peptide 13, with strong involvement of
peptide 12 in the epitope. Additional epitopes recognized by 184d
were centered at peptides 22, 33, 66, and 93. The third monkey
serum tested, 34, responded to this region of the CFA/I primary
structure, both at peptides 1, 12 and weakly at 14. Two other
epitopes were identified by 34, centered at peptides 67 and 128.
FIG. 26 illustrates the amino acids corresponding to the epitopes
of CFA/I as defined by the response of these three monkeys aligned
with the entire primary structure. The entire antigenic
determinants are mapped and areas of overlap criteria published by
Rothbard and Taylor [7]. The sequence numbers of the first amino
acid of the predicted segments are shown in Table 1.
Lymphocyte proliferation of monkey spleen cells to CFA/I synthetic
peptides. To determine which segments of the CFA/I protein are able
to stimulate proliferation of CFA/I immune primate lymphocytes in
vitro, three Rhesus monkeys were immunized with CFAI subunits, and
their splenic lymphocytes were cultured with synthetic overlapping
decapeptides which represented the entire CF/I sequence.
Concentrations of peptides used as antigen were 6.0, 0.6, and 0.6
ug/ml. Proliferative responses to the decapeptides were observed in
each of the three monkeys (FIGS. 9-11. The majority of the
responses occurred at the 0.6 and 0.06 ug/ml concentrations of
antigen and within distinct regions of the protein (peptides
beginning with residues 8-40, 70-80, and 27-137). A comparison of
the responses at the 6.0, 0.6 and 0.06 ug/ml concentrations
antigenic peptide for one monkey (2&2) are shown (FIGS. 12-14.
Taking into account all concentrations of antigen tested, spleen
cells from monkey 184D demonstrated a statistically significant
response to decapeptides beginning with CFA/I amino acid residues
3, 4, 8, 12, 15, 21, 26, 28, 33, 88, 102, 10, 133, 134, and 136
(FIG. 27. Monkey 34 had a significant response to decapeptides
beginning with residues 24, 31, 40, 48, 71, 72, 77, 78, 80, 87, and
102, 126 and 133 (FIG. 28); monkey 2Z2 responded to decapeptides
which began with residues 4, 9, 11, 12, 13, 14, 15, 16, 17, 20, 27,
35, 73, 79, 18, 127, 129, 132, and 133 (FIG. 27). Peptides
beginning with amino acid residues 3 through 2 were synthesized
with either a glutamic acid or an asparagine substituted for the
aspartic acid residue at position twelve to prevent truncated
peptides. The observed responses to peptides beginning with residue
8 (monkey 184d), and residues 9, 11, 12 (monkey 2Z2) occurred in
response to peptides that had the glutamic acid substitution.
However, the observed responses to peptides beginning with residue
3, 4, and 12 (monkey 184D), a well as residue 4 (monkey 2Z2)
occurred in response to peptides that had the asparagine
substitution. Monkey 34 did not respond to any of the peptides that
had the substitution at position twelve. All other responses shown
were to the natural amino acid sequence of the CFA/I protein.
Statistical significance was determined by comparing the cpm of
quadruplicate wells cultured with the CFA/I peptides to the cpm of
wells cultured with the CFA/I peptides to the cpm of wells cultured
with a control peptide.
Analysis of decapeptides that supported proliferation of
lymphocytes from CFA/I immune animals. Of the 39 different peptides
that supported proliferative responses, thirty contained a serine
residue, 19 contained a serine at either position 2, 3, or 4, and
nine had a serine specifically at position 3. Some of the most
robust responses were to the peptides that contained a serine
residue at the third position. The amino acid sequence of four such
peptides is shown in Table 3.
VII. DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered efficacious pharmaceutical compositions
wherein the relative amounts of antigen to the polymeric matrix are
within the ranges of 0.1 to 1.5% antigen (core loading) and 99.9 to
98.5% polymer, respectively. It is preferred that the relative
ratio between the lactide and glycolide component of the
poly(DL-lactide-co-glycolide) (DL-PLG) is within the range of 90:10
to 0:100. However, it is understood that effective core loads for
certain antigens will be influenced by its microscopic form (i.e.
bacteria, protozoa, viruses or fungi) and type of infection being
prevented. From a biological perspective, the DL-PLG or glycolide
monomer excipient are well suited for in vitro drug (antigen)
release because they elicit a minimal inflamatory response, are
biologically compatible, and degrades under physiologic conditions
to products that are nontoxic and readily metabolized.
Surprisingly, applicants have discovered an extremely effective
method for the protection against bacterial or viral infections in
the tissue of a mammal (human or nonhuman animal) caused by
enteropathogenic organisms comprising administering orally to said
animal an immunogenic amount of a pharmaceutical composition
consisting essentially of an antigen encapsulated within a
biodegradable polymeric matrix. When the polymeric matrix is
DL-PLG, the most preferred relative ratio between the lactide and
glycolide component is within the range of 48:52 to52:48. The
bacterial infection can be caused by bacteria (including any
derivative thereof) which include Salmonella typhi, Shigella
sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii,
Escheria coli, Vibro cholera, yersinia, staphylococcus, clostridium
and campylobacter. Representative viruses contemplated within the
scope of this invention, susceptible to treatment with the
above-described pharmaceutical compositions, are quite extensive.
For purposes of illustration, a partial listing of these viruses
(including any derivative thereof) include hepatitis A, hepatitus
B, rotaviruses, polio virus human immunodeficiency viruses (HIV),
Herpes Simplex virus type 1 (cold sores), Herpes Simplex virus type
2 (Herpesvirus genitalis), Varicella-zoster virus (chicken pox,
shingles), Epstein-Barr virus (infectious mononucleosis; glandular
fever; and Burkittis lymphoma), and cytomegalo viruses.
A further representation description of the instant invention is as
follows:
A. (1) To homogeneously disperse antigens of enteropathic organisms
within the polymeric matrix of biocompatible and biodegradable
microspheres, 1 nanogram (ng) to 12 microns in diameter, utilizing
equal molar parts of polymerized lactide and glycolide (50:50
DL-PLG, i.e. 48:52 to 52:48 DL-PLG) such that the core load is
within the range of about 0.1 to 1.5% by volume. The microspheres
containing the dispered antigen can then be used to immunize the
intestine to produce a humoral immune response composed of
secretory antibody, serum antibody and a cellular immune response
consisting of specific T-cells and B-cells. The immune response is
directed against the dispered antigen and will give protective
immunity against the pathogenic organism from which the antigen was
derived.
(2) AF/R1 pilus protein is an adherence factor that allows E. coli
RDEC-1 to attach to rabbit intestinal brush borders thus promoting
colonization resulting in diarrhea. AF/R1 pilus protein was
homogeneously dispered within a polymeric matrix of biocompatible
and biodegradable microspheres, 1-12 microns in diameter (FIG. 9
and photograph 1) using equal molar parts of polymerized lactide
and glycolide (50:50 DL-PLG) such that the core load was 0.62% by
weight.
(3) The microspheres were found to contain immunogenic AF/R1 by
immunizing both rabbit spleen (FIG. 10, and Peyer's patch (FIG. 3)
B-cells in vitro. The resultant cell supernatants contained
specific IgM antibody which recognized the AF/R1. The antibody
response was comparable to immunizing with AF/R1 alone.
(4) Microspheres containing 50 micrograms of AF/R1 were used to
intraintestinally (intraduodenally) immunize rabbits on two
separate occasions 1 week apart. One week later, compared to
rabbits receiving AF/R1 alone, the intestinal lymphoid tissue,
Peyer's patches, demonstrated an enhanced cellular immune response
to AF/R1 and to three AF/R1 linear peptide fragments 40-55, 79-94
and 108-123 by both lymphocyte transformation (T-cells) (FIGS. 12
and 13 and antibody producing B-cells (FIGS. 14 and 15. Similarly
enhanced B-cell responses were also detected in the spleen (FIGS.
16 and 17). An enhanced T-cell response was also detected in the
mesenteric lymph node and the spleen to one AF/R1 peptide fragment,
40-55 (FIGS. 18 and 19). The cellular immune response at two weeks
was too early for either a serum or secretory antibody response
(See Results in Table 1); but indicates that a secretory antibody
response will develop such that the rabbits so immunized could be
protected upon challenge with the E. coli RDEC-1.
B. Microspheres do not have to be made up just prior to use as with
liposomes. Also liposomes have not been effective in rabbits for
intestinal immunization of lipopolysaccharide antigens.
C. (1) Only a small amount of antigen is required (ugs) when
dispersed within microspheres compared to larger amounts (mgms)
when antigen is used alone for intestinal immunization.
(2) Antigen dispersed within microspheres can be used orally for
intestinal immunization whereas antigen alone used orally even with
gastric acid neutralization requires a large amount of antigen and
may not be effective for intestinal immunization.
(3) Synthetic peptides with and without attached synthetic
adjuvants representing peptide fragments of protein antigens can
also be dispersed within microspheres for oral-intestinal
immunization. Free peptides would be destroyed by digestive
processes at the level of the stomach and intestine. Any surviving
peptide would probably not be taken up by the intestine and
therefore be ineffective for intestinal immunization.
(4) Microspheres containing antigen maybe placed into gelatin-like
capsules for oral administration and intestinal release for
improved intestinal immunization.
(5) Microspheres promote antigen uptake from the intestine and the
development of cellular immune (T-cell and B-Cell) responses to
antigen components such as linear peptide fragments of protein
antigens.
(6) The development of intestinal T-cell responses to antigens
dispersed within microspheres indicate that T-cell immunological
memory will be established leading to long-lived intestinal
immunity. This long-lived intestinal immunity (T-cell) is very
difficult to establish by previous means of intestinal
immunization. Failure to establish long-lived intestinal immunity
is a fundamental difficulty for intestinal immunizaiton with
non-viable antigens. Without intestinal long-lived immunity only a
short lived secretory antibody response is established lasting a
few weeks after which no significant immunological protection may
remain.
D. (1) Oral intestinal immunization of rabbits against E. coli
RDEC-1 infection using either whole killed organisms, pilus protein
preparations or lipopolysaccharide preparations.
(2) Microspheres containing adherence pilus protein AF/R1 or its
antigen peptides for oral intestinal immunization of rabbits
against RDEC-1 infection.
(3) Oral-intestinal immunization of humans against enterotoxigenic
E. coli infection using either whole killed organisms, pilus
protein preparations or lipopolysaccharide preparations.
(4) Microspheres containing adherence pilus proteins CFA/I, II, III
and IV or their antigen peptides for oral intestinal immunization
of humans against human enterotoxigenic E. coli infections.
(5) Oral-intestinal immunization of humans against other enteric
pathogens as salmonella, shigella, camphlobacter, hepatitis-A
virus, rota virus and polio virus.
(6) Oral-intestinal immunization of animals and humans for mucosal
immunological protection at distal mucosal sites as the bronchial
tree in lungs, genito-urinary tract and breast tissue.
E. (1) The biocompatible, biodegradable co-polymer has a long
history of being safe for use in humans since it is the same one
used in resorbable suture material.
(2) By using the microspheres, we are now able to immunize the
intestine of animals and man with antigens not normally immunogenic
for the intestinal mucosa because they are either destroyed in the
intestine, unable to be taken up by the intestinal mucosa or only
weakly immunogenic if taken up.
(3) Establishing long-lived immunological memory in the intestine
is now possible because T-cells are immunized using
microspheres.
(4) Antigens that can be dispersed into microspheres for intestinal
immunization include the following: proteins, glycoporteins,
synthetic peptides, carbohydrates, synthetic polysaccharides,
lipids, glycolipids, lipopolysaccharides (LPS), synthetic
lipopolysaccharides and with and without attached adjuvants such as
synthetic muramyl dipeptide derivatives.
(5) The subsequent immune response can be directed to either
systemic (spleen and serum antibody) or local (intestine, Peyer's
patch) by the size of the microspheres used for the intestinal
immunization. Microspheres 5-10 microns in diameter remain within
macrophage cells at the level of the Peyer's patch in the intestine
and lead to a local intestinal immune response. Microspheres 1 ng-5
microns in diameter leave the Peyer's patch contained within
macrophages and migrate to the mesenteric lymph node and to the
spleen resulting in a systemic (serum antibody) immune
response.
(6) Local or systemic antibody mediated adverse reactions because
of preexisting antibody especially cytophyllic or IgE antibody may
be minimized or eliminated by using microspheres because of their
being phagocytized by macrophages and the antigen is only available
as being attached to the cell surface and not free. Only the free
antigen could become attached to specific IgE antibody bound to the
surface of mast cells resulting in mast cell release of bioactive
amines necessary for either local or systemic anaphylaxis.
(7) Immunization with microspheres containing antigen leads to
primarily IgA and IgG antibody responses rather than an IgE
antibody response, thus preventing subsequent adverse IgE antibody
reactions upon reexposure to the antigen.
In addition to the above, the encapsulation of the following
synthetic peptides are contemplated and considered to be well
within the scope of this invention:
(1) AF/R1 40-55;
(2) AF/R1 79-94;
(3) AF/R1 108-123;
(4) AF/R1 1-13;
(5) AF/R1 pepscan 16AA;
(6) CFA/I 1-13; and
(7) CFA/I pepscan 16AA.
(8) Synthetic Pepetides Containing CFA/I Pilus Protein T-cell
Epitopes (Starting Sequence # given)
4(Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro),
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu),
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
26(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro),
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser),
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln),
87(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe),
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr), and
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val), and mixtures
thereof.
(9) Synthetic Peptides Containing CFA/I Pilus Protein B-cell
(antibody) Eptiopes (Starting Sequence # given)
3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val),
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
22(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe),
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser),
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr),
127(GIy-Thr-Ala-Pro-Thr-AIa-Gly-Asn-Tyr-Ser), and
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof.
(10) synthetic peptides containing CFA/I pilus protein T-cell and
B-cell (antibody) epitopes (Starting Sequence # given)
3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro),
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and mixtures
thereof.
(11) synthetic peptides containing CFA/I pilus protein T-cell and
B-cell (antibody) epitopes (Starting Sequence # given)
CFA/I Pilus Protein T-cell Epitopes
4(Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro),
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu),
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
26(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro),
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser),
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln),
87(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe),
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr), and
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val); and synthetic
peptides containing CFA/I pilus protein Bell (antibody) epitopes
(Starting Sequence # given)
CFA/I Pilus Protein B-cell Epitopes
3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val),
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
22(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe),
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser),
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr),
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof.
(12) synthetic peptides containing CFA/I pilus protein T-cell and
B-cell (antibody) epitopes (Starting Sequence # given)
CFA/I Pilus Protein T-cell Epitopes
3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val),
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
22(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe),
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser),
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr),
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser); and
synthetic peptides containing CFA/I pilus protein T-cell and B-cell
(antibody) epitopes (Starting Sequence # given)
CFA/I Pilus Protein B-cell Epitopes
3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro),
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp),
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val),
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and mixtures
thereof.
We contemplate that the peptides can be used in vaccine constructed
for systemic administration.
EXAMPLES
The peptides in (8), (9), and (10) above can be made by classical
solution phase synthesis, solid phase synthesis or recombinant DNA
technology. These peptides can be incorporated in an oral vaccine
to prevent infection by CFA/I bearing enteropathogenic E. coli.
The herein offered examples provide methods for illustrating,
without any implied limitation, the practice of this invention in
the prevention of diseases caused by enteropathogenic
organisms.
The profile of the representative experiments have been chosen to
illustrate the effectiveness of the immunogenic polymeric
matrix-antigen composites.
All temperatures not otherwise indicated are in degrees Celcius
(.degree. C.) and parts or percentages are given by weight.
Materials and Methods
Animals. New Zealand White male rabbits were purchased from
Hazelton Research Products (Denver, Pa.), and were shown to be free
of current RDEC-1 infection by culture of rectal swabs. Animals
were 1-2 kg of body weight and lacked agglutinating anti-AF/R1
serum antibody at the time of the study.
Antigens. AF/R1 pili from E. coli RDEC-1 (015:H:K non-typable) were
purified by an ammonium sulfate precipitation method. The final
preparation migrated as a single band on SDS-polyacrylamide gel
electrophoresis and was shown to be greater than 95% pure by
scanning with laser densitometry when stained with coomassie blue.
Briefly, equal molar parts of DL-lactide and glycolide were
polymerized and then dissolved to incorporate AF/R1 into spherical
particles. The microspheres contained 0.62% protein by weight and
ranged in size from 1 to 12 micrometers. Both the microencapsulated
and non-encapsulated AF/R1 were sterilized by gamma irradiation
(0.3 megarads) before use.
Synthetic peptides (16 amino acids each) were selected by
theoretical criteria from the amino acid sequence of AF/R1 as
deduced from the nucleotide sequence. Three sets of software were
used for the selections. Software designed to predict B cell
epitopes based on hydrophilicity, flexibility, and other criteria
was developed by the University of Wisconsin Genetics Computer
Group. Software designed to predict T cell epitopes was based on
the Rothbard method was written by Stephen Van Albert (The Walter
Reed Army Institute of Research, Washington, D.C.). Software
designed to predict T cell epitopes based on the Berzofsky method
is published as the AMPHI program. The selected peptides were
synthesized by using conventional Merrifield solid phase
technology. AF/R1 40-55
(Thr-Asn-Ala-Cly-Thr-Asp-Ile-Gly-Ala-Asn-Lys-Ser-Phe-Thr-Leu-Lys)
was various dilutions of antigen and were incubated at 37.degree.
C. in 5% CO.sub.2. In other experiments, cultures were conducted in
a 24-well plates. In these experiments, 5.times.10.sup.6 cells were
cultured with or without antigen in a 2 ml volume. After 4 days,
100 microliters aliquots of cells were transferred to 96well plates
for pulsing and harvesting. Previous experiments have demonstrated
that optimal concentrations of antigen range from 150 ng/ml to 15
micrograms/ml in the 96-well plate assay and 1.5 ng/ml to 150 ng/ml
in the 24-well plate assay. These were the concentrations employed
in the current study. All cultures were pulsed with 1 Ci [.sup.3
H]thymidine (25 Ci/mmol, Amersham, Arlington Heights, Ill.) on day
4 of culture and were harvested for scintillation counting 6 hours
later.
Statistics. All cultures were conducted in replicates of four, and
standard deviations of the counts per minute (cpm) generally range
from 5-15% of the average cpm. In experiments where comparison of
individual animals and groups of animals is desirable, data is
shown as a stimulation index (SI) to facilitate the comparison. SI
were calculated by dividing the mean of cultures with antigen by
the mean of cultures without antigen (media control). Statistical
significance (p value) was determined by comparing the maximum
response for each antigen to the media control using the Student's
t test.
Results
Lymphocyte proliferation in response to protein and peptide
antigens of AF/R1. To determine if lymphoid tissues from AF/R1
immune animals respond in vitro to the antigens of AF/R1, the
immunity in a rabbit with preexisting high levels of anti-AF/R1
serum IgG was boosted twice by injection of 50 micrograms of
purified AF/R1 pili i.p. seven days apart. A week after the final
boost, in vitro lymphocyte proliferation of spleen and MLN cells
demonstrated a remarkable response to AF/R1 pili. In response to
the synthetic peptides, there was a small, but significant
proliferation of the spleen cells to all the AF/R1 peptides tested
as compared to cell cultures without antigen. Cells from the spleen
and Peyer's patches of non-immune animals failed to respond to
either AF/R1 or the synthetic peptides.
Microencapsulation of AF/R1 potentiates the mucosal cellular immune
response. To evaluate the effect that microencapsulation of AF/R1
may have on the cellular mucosal immune response to that antigen,
naive rabbits were primed twice with 50 micrograms of either
microencapsulated or non-encapsulated AF/R1 by endoscopic
intraduodenal inoculation seven days apart. All rabbits were
monitored daily and showed no evidence of clinical illness or
colonization by RDEC-1. One week following the last priming, the
rabbits were sacrificed and lymphoid tissues were cultured in the
presence of AF/R1 pili or peptide antigens. In rabbits which had
received non-encapsulated AF/R1, Peyer's Patch cells demonstrated a
low level but significant proliferation in vitro in response to
AF/R1 pili (FIG. 13), but not to any of the AF/R1 synthetic
peptides (FIGS. 14a-d). However, in rabbits which had received
microencapsulated AF/R1, Peyer's Patch cells demonstrated a
markedly enhanced response not only to AF/R1 (FIG. 13 but now
responded to the AF/R1 synthetic peptides 40-55 and 79-94 (FIGS.
14a and 14b). In addition, one of two rabbits primed with
microencapsulated AF/R1 (rabbit 135) responded to AF/R1 108-123,
but not AF/R1 40-47/79-86 (FIGS. 14c and 14d). In contrast, the
other rabbit in the group (rabbit 134) responded to AF/R1
40-47/79-86, but not to AF/R1 108-123 (FIGS. 14d and 14c).
Response of MLN cells to the antigens of AF/R1. Studies have shown
that cells undergoing blastogenesis in the MLN also tend to home
into mucosal areas, but experiments requiring in vitro lymphocyte
proliferation of rabbit MLN cells are difficult to conduct and to
interpret due to non-specific high background cpm in the media
controls. Our studies have shown that this problem can be avoided
by conducting the proliferative studies in 24-well plates, and then
moving aliquots of cells into 96-well plates for pulsing with
[.sup.3 H]thymidine as described in materials and methods. This
method of culture was employed for the remainder of the studies.
The MLN cells of all rabbits demonstrated a significant
proliferation in vitro in response to AF/R1 pili regardless of
whether they had been immunized with microencapsulated or
non-encapsulated AF/R1. However, only the rabbits which had
received microencapsulated AF/R1 were able to respond to the AF/R1
synthetic peptide 40-55 (FIG. 19). The MLN cells of rabbit 134 also
responded to AF/R1 79-94 (p<0.0001), AF/R1 108-123
(p<0.0001), and AF/R1 40-47/79-86 (p=0.0004); however, none of
the other rabbits demonstrated a MLN response to those three
peptides (data not shown).
Response of spleen cells to the antigens of AF/R1. Proliferative
responses of spleen cells to AF/R1 were very weak in all animals
tested (data not shown). However, in results which paralleled the
responses in MLN cells, there was a significant response to AF/R1
40-55 in rabbits which had been primed with microencapsulated AF/R1
(FIG. 20). There was no response to the other AF/R1 synthetic
peptides by spleen cells in either group of animals. The weak
response of spleen cells to AF/R1 provides further evidence that
these animals were naive to AF/R1 before the study began, and
indicates that the observed responses were not due to non-specific
stimulative factors such as lipopolysaccharide.
Summary
We have shown that there is an enhanced in vitro proliferative
response to both protein and its peptide antigens by rabbit Peyer's
patch cells following intraduodenal inoculation of antigen which
had been homogeneously dispersed into the polymeric matrix of
biodegradable, biocompatible microspheres. The immunopotentiating
effect of encapsulating purified AF/R1 pili as a mucosal delivery
system may be explained by one or more of the following mechanisms:
(a) Microencapsulation may help to protect the antigen from
degradation by digestive enzymes in the intestinal lumen. (b)
Microencapsulation has been found to effectively enhance the
delivery of a high concentration of antigen specifically into the
Peyer's patch. (c) Once inside the Peyer's patch,
microencapsulation appears to facilitate the rapid phagocytosis of
the antigen by macrophages, and the microspheres which are 5-10
micrometers become localized within the Peyer's patch. (d)
Microencapsulation of the antigen may improve the efficiency of
antigen presentation by decreasing the amount of enzymatic
degradation that takes place inside the macrophage before the
epitopes are protected by combining with Class II major
histocompatibility complex (MHC) molecules. (e) The slow,
controlled-release of antigen may produce a depot effect that
mimics the retention of antigen by the follicular dendritic cell.
(f) If the antigen of interest is soluble, microencapsulation
changes the antigen into a particulate form which appears to assist
in producing an IgA B cell response by shifting the cellular immune
response towards the T.sub.H and thereby not encouraging a response
by the T.sub.s. There is evidence that the GALT may be able to
discriminate between microbial and non-microbial (food) antigens in
part by the form of the antigen when it is first encountered, and
thus bacterial antigens do not necessarily have special antigenic
characteristics that make them different from food antigens, but
they are antigenic because of the bacterial context in which they
are presented. The particulate nature of microspheres may serve to
mimic that context. It may be important to note that we also
observed a significant response to AF/R1 in animals inoculated with
non-encapsulated pili; thus, some of this antigen which was still
in its native form was able to enter the Peyer's patch. This may be
explained by the fact that AF/R1 is known to mediate the attachment
of RDEC-1 to the Peyer's patch M-cell. If the antigen employed in
this type of study was not able to attach to micrometer M-cells,
one would expect to see an even greater difference in the responses
of animals which had received microencapsulated versus
non-encapsulated antigen.
The microspheres used in these experiments included a size range
from 1 to 12 micrometers. The 1 to 5 micrometer particles have been
shown to disseminate to the MLN and spleen within migrating
macrophages; thus, the observed proliferative responses by cells
from the MLN and spleen may reflect priming of MLN or splenic
lymphocytes by antigen-presenting/accessory cells which have
phagocytosed 1 to 5 micrometer antigen-laden microspheres in the
Peyer's patch and then disseminated onto the MLN. Alternatively,
these responses may be a result of the normal migration of antigen
stimulated lymphocytes that occurs from the Peyer's patch to the
MLN and on into the general circulation before homing to mucosal
sites. Proliferative responses by MLN cells are of interest because
it has been shown that cells undergoing blastogenesis in the MLN
tend to migrate onto mucosal areas. However, studies involving in
vitro lymphocyte proliferation of rabbit MLN cells can be very
difficult to conduct and to interpret due to non-specific high
background cpm in the media controls. By simultaneously conducting
experiments using different protocols, we have found that this
problem can be prevented by avoiding the use of fetal calf serum in
the culture and by initially plating the cells in 24-well plates.
Using this method, the blasting lymphocytes are easily transferred
to a 96well plate where they receive the [.sup.3 H]thymidine, while
fibroblasts and other adherent cells remain behind and thus do not
inflate the background cpm.
The proliferative response to the peptide antigens was of
particular interest in these studies. The rabbits that received
non-encapsulated AF/R1 failed to respond to any of the peptides
tested either at the level of the Peyer's patch, the MLN, or the
spleen. In contrast, Peyer's patch cells from the animals that
received microencapsulated AF/R1 responded to all the peptides
tested with two exceptions: Rabbit 134 did not respond to AF/R1
108-123, and rabbit 135 did not respond to AF/R1 40-47/79-86. The
reason for these non-responses is not clear, but it probably is not
due to MHC restrictions as evidenced by the fact that rabbit 134
was able to respond to AF/R1 108-123 at the level of the MLN. The
non-responses may be due to varing kinetics of sensitized T cell
migration in different rabbits, or they may reflect differences in
the efficiency of antigen presentation by cells from different
lymphoid tissues of these animals. Of all the synthetic peptides
tested, only AF/R1 40-55, (the one selected as a probable B cell
epitope), was recognized by serum from an AF/R1 hyperimmune rabbit.
In addition, this peptide was the only one that was uniformly
recognized by Peyer's patch, MLN, and spleen cells from both
rabbit. In addition, this peptide was the only one that was
uniformly recognized by Peyer's patch, MLN, and spleen cells from
both rabbits that were immunized with microencapsulated AF/R1. The
recognition by anti-AF/R1 serum antibodies indicates that the amino
acid sequence of this peptide includes an immunodominant B cell
epitope. Thus AF/R1 40-55 may readily bind to antigen-specific B
cells thereby leading to an efficient B cell presentation of this
antigen to sensitized T cells. Even though AF/R1 40-55 was not
selected as a probable T cell epitope by either the Rothbard or
Berzofsky methods, the current study clearly indicates that this
peptide can also stimulate a proliferative immune response.
Although further studies are required to definitively show that the
proliferating cells are indeed T cells, the responses observed in
this study are most likely due to the blast transformation of cells
from the lineage. Therefore, AF/R1 40-55 appears to contain a T
cell epitope in addition to the immunodominant B cell epitope, and
this area of the AF/R1 protein may thereby play an important role
in the overall immune response and subsequent protection against
RDEC-1.
The proliferative responses of spleen cells was low in all animals
tested; however, we feel tht this may be simply a matter of the
kinetics of cellular migration. The rabbits in this study were
sacrificed only two weeks after their first exposure to antigen.
This relatively short time period may not have provided sufficient
time for cells that were produced by Peyer's patch and MLN blasts
to have migrated as far as the spleen in sufficient numbers.
An ideal mucosal vaccine preparation would not only assist in the
uptake and presentation of the immunogen of interst, but it would
also be effective without requiring carrier molecules or adjuvants
which may complicate vaccine production or delay regulatory
approval. The incorporation of antigen into microspheres appears to
provide an ideal mucosal delivery system for oral vaccine
immunogens because the observed immunopotentiating effect is
achieved without the need for carriers of adjuvants. This ability
may prove to be of great value, particularly to enhance the
delivery of oral synthetic peptide vaccines to the GALT.
TABLE 10 Linear B-Cell Epitopes of CFA/I in Monkeys Sequence
Individuals Position Responding Consensus Site 1. 11-21 3 VDPVIDLLQ
2. 93-101 2 AKEFEAAA 3. 124-136 2 GPAPT 4. 66-74 2 PQLTDVLN 5.
22-29 2 GNALPSAV 6. 32-40 1 KTF* 7. 38-45 1 8. 3-11 1 *Overlap
between epitope 6 and 7
TABLE 11 Prediction of T cell epitopes within the CFA/I
molecule.sup.a Predicted Amphipathic Segments 7 aa blocks 11 aa
blocks Rothbard Criteria 22-25 8-11 16 34-38 32-44 30 40-46 51-71
38 50-53 86-92 44 56-62 102-108 57 64-71 130-131 61 104-108 135-137
70 131-137 116 124 127 137 .sup.a The sequence numbers of the first
amino acid of the predicted T cell epitopes are shown. Software
designed to predict T cell epitopes based on the Berzofsky method
was published as the AMPHI program. It predicts amphipathic amino
acid segments by evaluating 7 or 11 residues as a block #and
assigning a score to the middle residue of that block. Software
designed to predict T cell epitopes based on the Rothbard method
was written by Stephen Van Albert (The Walter Reed Army Institute
of Research, Washington, D.C.).
TABLE 11 Prediction of T cell epitopes within the CFA/I
molecule.sup.a Predicted Amphipathic Segments 7 aa blocks 11 aa
blocks Rothbard Criteria 22-25 8-11 16 34-38 32-44 30 40-46 51-71
38 50-53 86-92 44 56-62 102-108 57 64-71 130-131 61 104-108 135-137
70 131-137 116 124 127 137 .sup.a The sequence numbers of the first
amino acid of the predicted T cell epitopes are shown. Software
designed to predict T cell epitopes based on the Berzofsky method
was published as the AMPHI program. It predicts amphipathic amino
acid segments by evaluating 7 or 11 residues as a block #and
assigning a score to the middle residue of that block. Software
designed to predict T cell epitopes based on the Rothbard method
was written by Stephen Van Albert (The Walter Reed Army Institute
of Research, Washington, D.C.).
Demonstrative Evidence of Protective Immunity
RDEC-1 is an eteroadherent diarrhea producing E. coli in rabbit.
Its attachment to the mucosa is by the adhesin (AF/R1 pili). The
adhesin is an excellent vaccine candidate. It may intitiate a
mucosal response but is susceptiple to digestion in the gut. The
incorporation of AF/R1 into biocompabible, nondigestible
microspheres enhanced mucosal cellular immune respones to RDEC-1.
We have demonstrated that immunization with AF/R1 Pili in
microspheres protect rabbits against infection with RDEC-1.
Six rabbits received intra-duodenal immunizaiton of AF/R1
microspheres (0.62% coreloading by weight) at 200 ug AF/R1 on day 0
then boosted with 100 ug AF/R1 in microspheres on days 7, 14, and
21 followed by RDEC-1 challenge with 10.sup.8 organisms one week
latter than observed for 1 week and then sacrificed, unimmunized
rabbits were challenged with 10.sup.8 RDEC-1 only and observed 1
week than sacrified. Also, 2 rabbits were immunized only then were
sacrificed 10 days latter. Only one of these animals had bile IgA
antibodies to AF/R1. but both had specific sensitized T cells which
released IL-4 upon challenge in the spleen, Peyer's patch and
illeal lamina propria. All nine immunized animals developed
diarrhea and weight loss which was significant at the p<0.001
level compared to the immunized animals which displayed no diarrhea
and no weight loss. The immunized animals colonized the intestinal
tract with RDEC-1 the same as the unimmunized animals. However,
there was a striking difference regarding the adherence of RDEC-1
to the mucosa. No adherence was seen in cecum in the immunized
animals compared to 4/7 in the unimmunized side animals. This
difference was significant to the p<0.01 level. The RDEC-1
exposure although not producing disease in the immunized animals
did effect a booster immunization as relected in the increase in
anti-AF/R1 antibody containing cells in the muscosa similiar to the
immunized rabbits. This study clearly demonstrated complete
protection against RDEC-1 infection and strongly indicates similiar
results should be expected with entertoxigenicity E. coli using the
Colony Forming Antigens (CFA's) in microsphere vaccines.
Summary Statement of Protective Immunity Showings
RDEC-1 infection of rabbits causes an enteroadherent E. coli
diarrheal disease, and provides a model for the study of
adherence-factor immunity. Pilus adhesions are vaccine candidates,
but purified pili are subject to intestinal degradation. Previously
we showed potentiation of the mucosal cellular immune response to
the AF/R1 pilus of RDEC-1 by incorporation into biodegradable
polylactide-coglycolide microspheres (AF/R1-MS). We now present
efficacy testing of this vaccine. Six rabbits were primed with 200
ug and boosted with 100 ug of AF/R1-MS weekly .times.3, then
challenged at week 5 with 10.sup.8 CFU of RDEC-1 expressing AF/R1.
Nine unvaccinated rabbits were also challenged. Two rabbits
vaccinated with AF/R1-MS were sacrificed at week 5, without
challenge, for measurement of anti-AF/R1 antibodies in bile (by
ELISA) and anti-AF/R1 containing cells (ACC) in the intestinal
lamina propria (by immunohistochemistry). Attachment of RDEC-1 to
intestinal epithelial cells was estimated (0.4+) by
immunoperoxidase staining of histologic sections. Colonizaiton of
intestinal fluid was measured by culture of intestinal flushes.
Results: Rabbits given AF/R1-MS remained well and 4/6 gained weight
after challenge, whereas 9/9 unvaccinated rabbits lost weight after
challenge (mean weight change +10 vs -270 gms p<0.001), (see
FIG. 35). The mean score of RDEC-1 attachment to the cecal
epithelium was 0 in vaccinated, and 2+ in unvaccinated animals (see
FIG. 36). RDEC-1 colonizaiton (log CFU/gm) in cecal fluids was
similar in both groups (mean 6.3 vs 7.3; p=0.09) (see FIG. 34). ACC
were not seen in the lamina propria of vaccinated but unchallenged
animals, but anti-pilus IgA antibody levels in bile were increased
1 S.D. over negative controls in 1 animal. Conclusions: Vaccination
with AF/R1-MS was safe and protected rabbits against RDEC-1
disease. Protection was associated with interference with RDEC-1
adherence to the mucosal surface, but lumenal colonization was not
prevented.
More recently, applicants have focused on areas of this invention
related to an immunostimulating composition for the burst-free,
sustained, programmable release of active material(s) over a period
from 1 to 100 days, which comprises encapsulating microspheres,
which may contain a pharmaceutically-acceptable adjuvant, wherein
said microspheres are comprised of (a) a blend of uncapped and
end-capped biodegradable-biocompatible
poly(DL-lactide-co-glycolide) as the bulk matrix, wherein the
relative ratio between the amount of lactide and glycolide
components are within the range of 90:10 to 40:60 and the
poly(DL-lactide-co-glycolide) is a blend of uncapped and end-capped
forms in ratios ranging from 100:0 to 1 to 99, and (b) active
material such as an immunogenic substance comprising Colony Factor
Antigen (DFA/II, hepatitis B surface antigen (HBsAg)), and/or a
physiologically similar antigen that serves to elicit the
production of antibodies in a mammal (human or nonhuman).
These areas of invention are referred to herein after as Part II
and Part III, respectively, and are itemized as follows:
1. An immunostimulating composition for the burst-free, sustained,
programmable release of active material(s) over a period from 1 to
100 days, which comprises encapsulating-microspheres, which may
contain a pharmaceutically-acceptable adjuvant, wherein said
microspheres having a diameter between 1 nanogram (ng) to 10
microns (um) are comprised of (a) a blend of uncapped and
end-capped biodegradable-biocompatible poly
(DL-lactide-co-glycolide) as the bulk matrix, wherein the relative
ratio between the amount of lactide and glycolide components are
within the range of 90:10 to 40:60, and the
poly(DL-lactide-co-glycolide) is a blend of uncapped and end-capped
forms in ratios ranging from 100:0 to 1 to 99, and (b) active
material such as an immunogenic substance comprising Colony Factor
Antigen (CFA/II), hepatitis B surface antigen (HBsAg), and/or a
physiologically similar antigen that serves to elicit the
production of antibodies in a mammal (human or nonhuman).
2. An immunostimulating composition according to Item 1 wherein the
amount of said immunogenic substance is within the range of 0.1 to
1.5% based on the volume of said bulk matrix.
3. An immunostimulating composition according to Item 2 wherein the
relative ratio between the lactide and glycolide component is
within the range of 48:52 to 52:48.
4. An immunostimulating composition according to Item 2 wherein the
size of more than 50% of said microspheres is between 5 to 10 um in
diameter by volume.
5. A vaccine comprising an immunostimulating composition of Item 4
and a sterile, pharmaceutically-acceptable carrier therefor.
6. A vaccine comprising an immunostimulating composition of Item 5
wherein said immunogenic substance is Colony Factor Antigen
(CFA/II).
7. A vaccine comprising an immunostimulating composition of Item 5
wherein said immunogenic substance is hepatitis B surface antigen
(HBsAg).
8. A method for the vaccination against bacterial infection
comprising administering to a human, an antibactericidally
effective amount of a composition of Item 6.
9. A method according to item 7 wherein the bacterial infection is
caused by a bacteria selected from the group consisting essentially
of Salmonella typhi, Shigella Sonnei, Shigella Flexneri, Shigella
dysenteriae, Shigella boydii, Escheria coli, Vibrio cholera,
yersinia, staphylococus, clostridium, and campylobacter.
10. A method for the vaccination against viral infection comprising
administering to a human an antivirally effective amount of a
composition of Item 7.
11. A diagnostic assay for bacterial infections comprising a
composition of Item 4.
12. A method of preparing an immunotherapeutic agent against
infections caused by a bacteria comprising the step of immunizing a
plasma donor with a vaccine according to Item 6 such that a
hyperimmune globulin is produced which contains antibodies directed
against the bacteria.
13. A method preparing an immunotherapeutic agent against
infections caused by a virus comprising the step of immunizing a
plasma donor with a vaccine according to Item 7 such that
hyperimmune globulin is produced which contains antibodies directed
against the hepatitis B virus.
14. An immunotherapy method comprising the step of administering to
a subject an immunostimulatory amount of hyperimmune globulin
prepared according to Item 12.
15. An immunotherapy method comprising the step of administering to
a subject an immunostimulatory amount of hyperimmune globulin
prepared according to Item 13.
16. A method for the protection against infection of a mammal
(human or nonhuman animal) by enteropathogenic organisms or
hepatitis B virus comprising administering to said mammal an
immunogenic amount of an immunostimulating composition of Item
3.
17. A method according to Item 16 wherein the immunostimulating
composition is administered orally.
18. A method according to Item 16 wherein the immunostimulating
composition is administered parenterally.
Part II
In sum, the Colony Factor Antigen (CFA/II) from enterotoxigenic E
coli (ETEC) prepared under GMP was successfully incorporated into
biodegradable polymer microspheres (CFA/II BPM) under GMP and found
to be safe and immunogenic when administered intra-duodenally to
rabbits. CFA/II was incorporated into poly
(D,L-lactide-co-glycolide) (PLGA) microspheres which were
administered by direct endoscopy into the duodenum. Following
vaccination, Peyer's patchcells responded by lymphocyte
proliferation to in vitro challenge with CFA/II indicating the
CFA/II BPM to be immunogenic when administered intra-intestinally.
Also, B cells secreting specific anti CFA/II antibodies were found
in spleens following vaccination. No pathological changes were
found following total necropsies of 10 rabbits vaccinated with
CFA/II BPM. As a potency test, high serum IgG antibody titers to
CFA/II were produced following intra-muscular administration of
CFA/II BPM to additional rabbits. The CFA/II BPM contained 63%
between 5-10 um by volume particle size distribution; 1.17% protein
content; 2.15% moisture; <0.01% acetonitrile; 1.6% heptane; 22
nonpathogenic bacteria and 3 fungi per 1 mgm protein dose; and
passed the general safety test. We conclude that the CFA/II BPM
oral vaccine is immunogenic and safe to begin a Phase I clinical
safety study following IND approval.
Introduction
Enterotoxigenic Escherichia coli (ETEC) causes diarrheal disease
with an estimated 650,000,000 cases anually in developing countries
resulting in 500,000 deaths predominantly in the pediatric age
groups. Currently there is no vaccine against ETEC induced
diarrhea. The availability of an effective oral vaccine would be of
great value to the people of South America, Africa and and Asia as
well as the millions of people who travel to these high risk areas
and account for half of the annual cases.
The first step in pathogenesis is adherence to the small intestine
epithelial cells by protein fimbrial (pilus) adhesins called
colonization factor antigen (CFA). Three major CFAs have been
recognized, CFA/I, CFA/II and CFA/IV. (25)
Ten human volunteers who were immunized orally twice weekly for 4
weeks with CFA/II developed a poor antibody response and did not
show any significant protection when challenged with pathogenic
ETEC (26). This disappointing response was attributed to adverse
effects of gastric acid, even at neutral pH, of fimbrial proteins
(27). When the vaccine was administered by inoculation directly
into the duodenum, 4 of 5 immunized volunteers developed a
significant rise in secretory IgA with CFA/II antibody (26).
D and L-lactic acid and glycolic acid, as homo- and copolymers, are
biodegradable and permit slow and continued release of antigen with
a resultant adjuvant activity. These polymers have been shown to be
safe in a variety of applications in human beings and in animals
(28-32). Delivery of antigens via microspheres composed of
biodegradable, biocompatible lactide/glycolide polymers (29-32) may
enhance the mucosal response be protecting the antigen from
digestion and targeting them to lymphoid cells in Peyer's patches
(29-32). McQueen et al. (33) have shown that E coli AF/R1 pili in
PLGA microspheres, introduced intra-duodenally in rabbits,
protected them against diarrhea and weight loss when challenged
with the parent strain rabbit diarrheagenic strain of E coli
(RDEC-1). Only one vaccinated rabbit of six lost weight and only
one had soft pelleted stool. In contrast, all control unvaccinated
animals became ill, lost weight, and shed soft pellets or unformed
mucoid stool. Significant lymphocyte proliferation to AF/R1 from
Peyer's patches and ordinary IgA anti AF/R1 antibody levels were
seen.
In order to improve the CFA/II vaccine it was incorporated into
PLGA microspheres under GMP in order to protect it from digestion
and target it to the intestinal lymphoid system. The CFA/II BPM
vaccine has undergone pre-clinical evaluation and has been found to
be safe and immunogenic.
Materials and Methods
Preparation of CFA/II Pilus Vaccine. Under Good Laboratory and Good
Manufacturing Practices, E. coli. strain M424C1-06;816 producing
CFA/II were cultured in 75-80 CFA agar plates (24.times.24 cm) for
24 hrs then harvested by scraping. The harvest was homogenized at
slow speed for 30 minutes with over head drive unit and cup
immersed in an ice bath. The homogenate was centrifuge at 4.degree.
C. at 16, 500.times.g for 30 minutes. The supernatant saved and the
pellet rehomogenized and centrifuged with the supernatants pooled.
The supernatant pool was centrifuged at 50,000.times.g for 45
minutes. The supernatant treated with ammonium sulfate at 20%
satuaration, stirred 30 minutes at 4.degree. C. than stored at
4.degree. C. for 16 hrs then centrifuged at 19,700.times.g for 30
minutes. The supernatant saved and treated with ammonium sulfate at
45% saturation, stirred 30 minutes at 4.degree. C., stored at
4.degree. C. for 66-72 hrs, then centrifuged at 19,700.times.g for
45 minutes. The pellet was resuspended in about 100 mls of PBS
containing 0.5% formalin and held at 22.degree. for 18 hrs then
dialyzed for 45-50 hrs against PBS at 4.degree. C. using a total of
12 liters in 2 liter amounts. The dialysis was terminated when the
PBS contained less then 0.03% formalin using Nessler's reagent and
fuchsin sulfuose acid reagent. The final product contained 1 mgm
protein/ml PBS, was sterile and passed the general safety test.
Preparation of Desalted CFA/II Vaccine. Two ml of the CFA/II
vaccine were placed into a Centricon 30 tube and centrifuged at
1700 rpm at 4-6.degree. C. (Beckman model GPR centrifuge equipped
with GA-24fixed angle rotor) until all the buffer solution passed
through the filter (about 90-120 minutes). Sterile water was added
to each tube to disperse the CFA/II retained on the filter. The
desalted antigen dispersions from all tube were pooled and then
divided into five equal parts by weight so as to contain 20 mg of
the CFA/II each. The desalted antigen dispersion was stored at -10
to -20.degree. C.
Freeze Drying of the Desalted CFA/II Dispersion. 80 mg of sucrose
was added to each part of the CFA/II dispersion. The resulting
mixture was flash-frozen using a dry ice-acetone bath (100-150 ml
od acetone and 50-100 g of dry ice). The frozen solution was freeze
dried overnight using Repp Sublimator 16 freeze dryer at vacuum of
1 micrometer of mercury and a shelf temperature not exceeding
37.degree. C.
CFA/II Biodegradable Polymer Microspheres
Particle size distribution. About 1 mgm of microspheres were
dispersed in 2 ml of 1% Polysorbate 60.degree. (Ruger Chemical Co.
Inc. Irvington, N.J.) in water in a 5 ml capacity glass vial by
sonication. This dispersion was observed under a calibrated optical
microscope with 43.times.magification. Using a precalibrated
eye-piece micrometer, the diameter of 150 randomly chosen
microspheres, was determined and the microsphere size distribution
was determined
Scanning Electron Microscopic Analysis. Microspheres were sprinkled
or the surface of 10 mm stub covered with a non-conductive adhesive
(Sticky-Tab, Ernest F. Fullem, Inc., Lutham, N.Y.) Samples were
coated with gold/palladium in an automatic sputter-coating
opparatus (Samsputter-2A, Tonsimis Research Corporation). The
samples were examined with a Hitachi S-450 scanning electron
microscope operated at 15-20 KV.
Preparation of CFA/II Microspheres. Solvent extraction techique was
used to encapsulate the freeze dried CFA/II into
poly(lactide-co-glycolide)(Medisorb Techologies International,
visocity 0.73 dl/g) microspheres in the 1-10 um size range to
achieve theoretical antigen loading of 1% by weight. The freeze
dried antigen-sugar & matrix was dispersed in an acetolnitrile
solution of the polymer and then emulsified to achieve desired
droplet size. Microspheres were solidified and recovered by using
heptane as extracting solvent. The microsphere batches were pooled
and vacuum dried to remove traces of solvent.
Protein Content. The CFA/II microspheres were dissolved in 0.9% SDS
in 0.1N NaOH for 18 hr with stirring then neutralized to pH 7 and
assayed. The micro bicichoninic acid (BCA) method was utilized with
both lactic acid and glycolic acid blanks and compared to bovine
serum albumin (BSA) standard and results expressed as percent by
weight.
Moisture Content. One hundred and fifty mgm of CFA/II microspheres
were dissolved in 3 ml of acetonitrile by sonication for 3 hours.
One ml sample was injected into a Karl Ficher titrimeter and triter
reading observed was recorded and acetonitrile blank was
substracted to determined percent water content.
Acetonitrile and Heptane Residuals. Ten mgm of CFA II microspheres
were dissolved in 1 ml DMF then analysed using gas chromatography
and comparing peak heights to external standards of either
acetonrile or heptane diluted in DMF with 10 mgm of blank
microspheres. The results are expressed as percent by weight.
Microbial load. One hundred mgm of CFA/II microsphere(single dose)
are suspended in 2 ml of sterile saline than poured into 2 blood
agar plates (1 ml each). All colonies are counted and identified
after 48 hours in culture at 37.degree. C. and expressed as total
number. Similiar amount of microspheres is in 0.25 ml aliquots
poured onto 4 different fungal culture plates (Sabhiragar, casein
peptone agar with chloramphenicol, brain heart infusion agar with
chloramphenol and genimycin or chloramphenicol alone) and cultured
at 30.degree. for 5 weeks and the colories counted & identified
and expressed as total number.
CFA/II Release From Microsphere Study. Thirty mgm samples in
triplicate were placed in 2 ml conical upright microcentrifuge
tubes containing 1 ml of PBS at pH 7.4. The tubes were capped and
kept immerized in a water bath maintained at 37.degree. C. with
constant agitation. The samples were withdrawn at 1, 3, 6, 8, 15
and 22 hour time intervals by centrifuging the sample tubes for 5
minutes at the maximum speed of bench top centrifuge. The release
medium was collected through a 5 um nylor screen for CFA/II protein
analysis using the micro BCA method and comparing results to BSA
standard and expressing results as percent cumulative release of
CFA/II.
General Safety Test. Two doses of one hundred mgm CFA/II
microspheres were suspended by sonication for 5 minutes in 3.1 mls
of sterile vaccine dilutent consisting of injectable saline
containing 0.5% Polysorbate 60.sup.R N.F., 0.03 ml were injected
intraperitoneally into each of 2 mice and 3 mls were administered
by gastric lavage to each of 2 guinea pigs. The animals were
weighed both before and at 7 days following the vaccine
administration. All animals were observed daily for any signs of
toxicity.
Rabbits. 1.5-2 kilogram male specific pathogen free New Zealand
white rabbits, obtained from closed colony maintained at the
National Institute of Health, Bethesda, Md. They were selected for
study if they did not have measurable serum antibodies at 1:2
dilution to CFA/II antigens by ELISA and were not colonized by E.
coli as determined by culture of rectal swabs.
Intra-Muscular Immunization of Rabbits and ELISA. Two Rabbits were
immunized with CFA/II microsphere vaccine at 25 ug protein in two
different sites intramuscularly on day 0. Sera were obtained from
all animals before immunization on day o and days 7 and 14. The
sera were tested by ELISA for IgG antibodies to CFA/II antigen and
individual coli surface (CS) proteins CS3 and CS1. ELISA plates
were coated with 3 ug/ml of either CFA/II antigen, CS3 or CS1
protein (150 ul/well) and incubated with 150 ul/well of PBS with
0.1% BSA for four hours at room temperature. The PBS with 0.1% BSA
is washed out with PBS and 100 ul/well of different dilutions of
each rabbit serum in triplicate was added to the plates. The
dilutions ranged from undiluted to 1:1,000,00. The plates were
incubated with the sera for 3 hours at 37.degree. C. The sera were
washed out with PBS and then horse radish peroxidase-conjugated
goat anti-rabbit IgG was added to the plates at a 1:1000 dilution
(100 ul/well). The plates were incubated for 1 hour at room
temperature with the peroxidase conjugate. The conjugates were
washed out of the plates with PBS and 100 ul/well of an ABTS
substrate solution (Kikegaard and Perry Laboratories) was added to
each well in the plates. The plates were read using the ELISA
reader(Dynatech Laboratories MR 580) at a wave length of 405 nm
after 15 minutes. The results are measured and expressed as
antibody titers.
Intra-duodenal Vaccination of Rabbits. Rabbits (N=5) were
vaccinated with CFA/II microspheres containing either 25 or 50 ug
of protein suspended in 1 ml of PBS containing 0.5% Polysorbrate
60.sup.R on day 0 and 7 by sonication. The microspheres were
injected through an Olympus BF type P10 bronchoscope into the
duodenum of the rabbits following sedation with an intra muscular
injection of ketamine HCl (50 mgm I.M.)(Ketaset, Fort Dodge
Laboratories, Fort Dodge, Iowa) and Lylazine (10 mgm I.M.) (Rompom
Malay Corporation, Shnanee, Kans.). The endoscope was advanced
ready under direct vission into the stomach which was insulated
with a 50 ml bolus of room air via a catheter passed through the
biospy channel. The catheter was advanced through the pylorus 3-4
cm into the duodemum and the microsphere suspension in 1 ml of PBS
was injected, followed by a 9 ml flush of PBS and removal of the
air bolus. The rabbits were sacrified by chemical euthanasia at day
14.
Anti-CFA/II Stimulated Lymphocyted Transformation. The Peyer's
Patchs were removed and cell suspension obtained by teasing and
irigation with a 20 guage needle and syringe. The cells were placed
in 2 ml of media at a concentration of 2.5.times.10.sup.6 cells/ml
for each well of a 24 well plate. These cells were challenged
separately with BSA and the CFA/II antigen at doses of 500, 50 and
5 ng/ml in triplicate wells. The plates were incubated at
37.degree. C. with 5% CO.sub.2. On day 4 the cells were mixed while
still inside the wells and 100 ul were transferred into each of 4
wells in a 96 well flat bottom microculture plate. Thus, the
challenge at each antigen dose represented by 3 wells in the 24
well plate is now represented by 12 wells in the 96 well plate.
After the cells have been transferred, each well is pulsed with 20
ul of 50 uCi/ml tritiated thymidine. These pulsed plates were
incubated for 6 hrs then harvester with 96 Mach II Cell harvested
(Tourtec, Inc.). The lymphocyte proliferation was determined by the
tritriated thymidine incoporation measured in kilo counts per
minute (Kcpm) using the 1205 Beta Plate Liquid scintillation
counter (LKB, Wallac, Inc.). The results are expressed as mean
Kcpm.+-.SD and compared to media controls.
Anti-CFA/II Antibody Secreting B Cells. Spleen cells were obtained
from immunized rabbits on day 14 following intra-duodenal
immunization with CFA/II microsphere vaccine. The cells were placed
in 96 well round bottom microculture plate at a final concentration
of 6.times.10.sup.5 cells/well and incubated for 0, 1, 2, 3, 4 and
5 days at 37.degree. C. with 5 CO.sub.2. 96 well flat bottom
microculture plates were coated with 3 ug/ml of CFA/II antigen
overnight blocked with PBS with 0.05% Polysorbate 60.sup.R. On the
harvest days, the cells were gently flushed out of the wells of the
round bottom plates and transferred to the corresponding well in
the antigen coated, 96 well flat bottom microculture plates to be
tested for the presence of antibody secreting cells using ELISPOT
technique. The plates were incubated with the cells overnight at
4.degree. C. The cells were then washed out of the flat bottom
plates with PBS, and 100 ul/well of horserudish-peroxidase
conjugated, goat anti-rabbit total antibody (IgM, IgG, and IgA) at
a 1:1000 dilution were added to the plates. The Plates were
incubated for 1 hour at room temperature, at which time, the
conjugate was washed out of the plates with PBS. 0.1 mgm of agarose
was dissolved in 10 ml of PBS by boiling. After the agar solution
cooled but not hardened, 6 mgm of 4-chloro-naphthol, 2 mls of
methanol and 30 ul of hydrogen peroxide were added to make the
substrate solution. The solution was placed into the flat bottom
plates (100 ul/well) and the plates were held at 4.degree. C.
overnight so the agar could harden. The number of browish spots per
15 wells (total of 9.times.10.sup.6 spleen cells) was counted and
represents the number of antibody secreting cells per
9.times.10.sup.6 spleen cells.
Pathological Evaluation. Rabbits were euthanized by parenteral
overdose of sodium pentobarbital and were subjected to complete
necropsy. Sample of tissue including small and large intestine with
gut associated lymphoid tissue, spleen, mesenteric and mediastinal
lymph nodes, lung, trachea, liver and kidney were fixed by
immersion in 10% neutral buffered formalin. Tissues were routinely
processed for light microscopy and embedded in paraffin. Five
micron thick sections were stained with hematoxylin and eosin.
Statistical Analysis. The paired student t-test was used to
determine p values.
Results
Particle Size Distribution. The results of size frequency analysis
of 150 randomly chosen microspheres are shown in (FIG. 37). The
particle size distribution is plotted in % frequency against
particle size in diameter (size) expressed in um. The average
number frequency diameter is 4.6 um. The average volume frequency
diameter is 4.6 um. The percent volume between diameters of 5-10 um
is 63% and the percent volume less than 10 um diameter is 88%.
Scanning Electoron Microscopy. The microspheres are seen in (FIG.
38) which is a scanning electron photomicrograph. Nearly all the
microspheres are less than 10 um as compared to the 5 um bar. Also
the surfaces of the microsphere are smooth and demonstrate lack of
pores.
Protein Content. The protein loads of the individual batches are
the following: K62A8, 1.16%.+-.0.10 SD; K63A8, 1.023% .+-.0.17SD;
K64A8, 1.232%.+-.0.13 SD; and K65A8, 0.966%.+-.0.128 SD. The mean
average protein load is 1.16%.+-.0.15 SD. The protein load of the
CFA/II microsphere vaccine in the final dose vial is the following:
Lot L74F2, 1.175%.+-.0.17SD.
Moisture Content. The CFA/II microsphere vaccine (Lot 74F2) percent
water content was found using the Karl Fischer titrimeter method to
be 2.154% using triplicate samples.
Acetonitrile and Heptane Residuals. The acetonitrile residuals of
the 4 individual CFA/II microsphere batches are the following:
K62A8, <0.1%; K62A8, <0.1%, K64A8, <0.1%; and K65A8,
<0.1%. The acetonitrile residual of the CFA/II microsphere
vaccine in the final dose vial is the following: Lot L74F2,
0.07.+-.0.05%. The heptane residual of the 4 individual CFA/II
microsphere batches are the following:K62A8, 1.9%; K63A8, 1.4%;
K64A8, 1.6% and K65A8, 1.6%. Following pooling in heptane and
subsequent drying, the heptane residual of the CFA/II microsphere
vaccine in the final dose vial is the following: Lot L74F2,
1.6.+-.0.1%.
Microbial load. One hundred milligrams (a single dose) of CFA/II
microsphere vaccine (Lot L74F2) in the final dose vial was
suspended in a 2 ml of sterile saline and 1 ml poured onto a blood
agar culture plate.times.2. Twenty two colonies grew after 48 hours
of culture and 21 were identified as coagulase negative
staphlycoccus and 1 as a micrococus species. All these bacteria are
considered to be nonpathogenic to humans. An additional 100 mgms of
CFA/II microsphere vaccine (Lot L74F2) were suspended in 2 ml of
sterile saline and 0.25 ml poured onto four different fungal
culture agars and cultered for 5 weeks. Three fungal colonies grew
and each was identified as A. glaucus.
CFA Release From Microsphere Study. Three thirty mgm samples were
incubated each in 1 ml of PBS, pH 7.4 at 37.degree. C. for 0, 1, 3,
6, 8, 15 and 22 hours. The superanates were removed and replaced at
these times. The protein content was determined for each supernate
sample and the results are seen in (FIG. # 39). The results are
plotted as percent release of CFA/II against time in hours. An
average of 8% of CFA/II is released at one hour rising to 20% at 8
hours then a slower release to 25% at 22 hours.
General Safety Test. Two one hundred milligrams(a single dose) of
CFA/II microsphere vaccine in the final dose vials were suspended
in 3.1 mls of the sterile dilulent consisting of 0.85 N saline
prepared for injection plus Polysorbrate 60.sup.R at 0.5%. Two
Swiss mice (16.5 gm) were injected intraperitoneally with 0.03 mls
and two Hartley guinea pigs (350 gm) were administered by gastric
lavage 3.0 mls.
None of these animals displayed any signs of toxicity for 7 days.
The mice gained and average of 2.3 gms and the guinea pigs gained
and average, of 43 grams. The CFA/II microsphere vacccine therefore
passed the general safety test.
Serum IgG Antibody Responses. Two rabbits were immunized in two
separate sites intramuscularly with 25 ug of protein of CFA/II
microsphere vaccine (Lot L74F2) in the final dose vial. Sera
samples were obtained before and 7 and 14 days following
immunization. The IgG antibody titers to CFA/II CSI and CS3 protein
were determined using ELISA and the results seen in (FIG. 32). The
results are expressed as mean antibody titers against the different
antigens at 0, 7 and 14 days. High antibody titers greater than
1000 were seen at 7 days to both CS1 and CS3 protein which rose to
greater than 10,000 by day 14. The individuals titers to CFA/II are
seen in (FIG. 33). Rabbit 109 developed an antibody titer of 1,000
by day 7 rising to 3,000 by day 14. Rabbit 108 had a log higher
rise at day 7 and 2 log higher rise at day 14 being
3.times.10.sup.4 at day 7 going to 1.times.10.sup.5 at day 14.
Anti-CFA/II Stimulated Lymphocyte Transformation. Five rabbits were
immunized intra-duodenally with CFA/II microspheres containing
either 25 ug of protein (human dose equivalent) or 50 ug of protein
on days 0 and 7 and then sacrificied on day 14. The Peyer's patch
lymphocytes were challenged in vitro with CFA/II antigen, BSA media
and alone. The lymphocyte transformation was determined by
tritriated thymidine incorporation. The results of the high dose
immunization are seen in (FIG. 34). The results are expressed as
Kcpm against antigen dose. No response to BSA or media control is
seen in any of the five rabbits. All rabbits responded by
lymphocyte transformation in a dose dependent manner to the
CFA/II.
The highest dose responses were 3-10X's the media control are
highly significant with a p value of <0.002. The results of the
5 rabbits receiving the low dose immunization are seen in (FIG.
35). Rabbit #80 gave no response probably due to poor Peyer's patch
cell population which did not respond were to Conconavallin A
mitogenic stimulation either. The remaining 4 rabbits gave positive
responses with the high CFA/II dose response being 2-8.times.media
control and highly significant with p values of <0.009. Again no
response were seen to BSA compared to the media cont.
Anti-CFA/II Antibody Secreting B-Cells Five rabbits immunized
intraduodenally with CFA/II microsphere containing 50 ug of CFA/II
protein at days 0, 7 than sacrified at day 14 were studied. The
spleen cells were placed into microculture then ELISPOT forming
B-Cells secreting specific anti CFA/II antibody determined at days
0, 1, 2, 3, 4 and 5. The results are seen in (FIG. 36) and
expressed as # of antibody secreting cells per 9.times.10.sup.6
spleen cell against culture days. Positive responses were seen in
all 5 rabbits on days 2-5. Days of maximum responses occurred on
day 3 for rabbits 65 and 66; day 4 for rabbit 85; amd day 5 for
rabbits 83 and 86. The responses are highly significant being 7-115
times higher than the 1-2 cells seen on all days in 4 control
rabbit (67, 69, 72, 89) (FIG. 45). Here is a composite graph
expressing the mean counts.+-.ISD for all days of culture.
Pathological Evaluation. A consistent finding in the spleens of all
rabbits both the 25 and 50 ug protein dose groups was minimal to
mild diffuse lymphocytic hyperplasia the periarteriolar lymphatic
sheaths (T cell dependent areas). Two of five rabbits of the 50 ug
dose group (#83 and #86) also had mild lymphocytic hyperplasia of
splenic follicular (B cell dependent) areas. The three rabbits in
an untreated control group had histologically normal spleens.
Reactive hyperplasia of mesenteric lymph nodes was often seen in
vaccinated rabbits. Two of five rabbits in the 25 ug dose
equivalent group (#83 and #86) also had minimal to mild lymphocytic
hyperplasia of cortical follicular (B cell dependent) areas. The
mesenteric lymph nodes of the other vaccinated rabbits and of the
untreated control rabbits were within normal limits. Incidental or
background lesions found in one or more rabbits of all three group
were acute minimal to mild pnuemonia and foreign body
microgranulomas of the cecal gut associated lymphoid tissue.
Disscussion
McQueen et al (33) has found that the AF/R1 adhesin of rabbit
diarrheagenic Escherichai coli (RDEC-1) incorporated into
biodegrable microspheres could function as a safe and effective
oral intestinal vaccine in the rabbit diarrhea model. The AF/R1 was
incorporated into poly D,L-lactide-co-glycolide) microspheres and
administered intraduodenally. Jarboe et al (34) reported that
Peyer's patch cells obtained from rabbits immunized
intra-duodenually with AF/R1 in microspheres responded with
lymphocte proliferation upon in vitro challenge with AF/R1. This
early response at 14 days gave a clear indication as to the
immunogenicity of E. coli pili contained within the polymer
microspheres.
In developing an effective oral vaccine against enterotoxigenic E.
coli, CFA/II pili given as an oral vaccine was found to be
ineffective. The CFA/II pilus proteins were found to be rapidly
degraded when treated with 0.1 mHCl and pepsin conditions mimicking
those contained in the stomach (27). The CFA/II was found to be
immunogenic when given in high doses intraintestinally producing
intestinal secretary IgA antibodies (26).
The CFA/II vaccine has now been incorporated into poly(D,L
lactide-co-glycolide) microspheres under Good Manufacturing
Practices and tested under Good Laboratory Practices. The
microspheres, are spherical, smooth surfaced and without pores. The
majority (63%) are between 5-10 um in diameter by volume. This size
range has been suggested to promote localization within the Peyer's
patch in mice and perhaps enhance local immunization (29-32). The
protein content being 1.174% is close to 1% which was the goal of
the vaccine formulation. One percent was chosen because 0.62% was
the core loading of the AF/R1 microspheres which were effective.
Also a small precentage perhaps 1-5% (35) is anticipated to be
taken up from the intestine, a higher protein content would lead to
considerable loss of protein.
The organic residuals are of course a concern. Heptane exposure
would be 1.7 mgm per vaccine dose. This is compared to the
occupational maximum allowable exposure of 1800 mgm/15 min.
Therefore, the heptane contained with the CFA/II microsphere
vaccine appears to be a safe level. The acetonitrile is very low
-0.1 mgm per vaccine dose. The human oral TDLO is 570
mgm.backslash.Kg (any non letheal toxicity). Therefore, the
acetonitrile contained with the CFA/II microsphere vaccine appears
to be at a safe level. The CFA/II vaccine was produced under
sterile conditions. However, the process of incorporation of the
desalted CFA/II vaccine into the polymer
The antibody secreting B-cells demonstrated in the rabbit spleen at
14 days is a clear indication that B-cells have been immunized.
They may represent resident B-cells immunized in the spleen or
B-cells immunized at the level of the Peyer's patches and are
migrating through the spleen to return to the intestial mucosal
lamina propria (1-3). The delay of several days before secreted
antibody is detected suggests either manuration is required of the
B-cells or that down regulation may be present initially and lost
with time in culture.
Further evidence of immunization by the CFA/II microsphere vaccine
given intra-duodenually is demonstrated by the lymphatic
hyperplasia in the spleen seen to a greater extend in the rabbits
receiving the lower dose 5/5 compared to 2/5 of the rabbits
receiving the higher 50 ug protein dose. On the other hand, greater
T-cell dependent area lymphatic hyperplasia in the mesenteric lymph
nodes were seen in rabbits receiving the higher 50 ug dose, 4/5
compared to 2/5. These changes are most likely due to the vaccine
since similar changes were not seen in three untreated control
rabbits. Also no abnormal pathological changes attributable to the
vaccine were seen.
The CFA/II BPM vaccine has undergone pre-clinical evaluation and
has been found safe and immunogenic. This vaccine is ready for
clinical Part I safety testing following FDA's IND approval.
Part III
In sum, alum precipitation, vaccination regimen and controlled
delivery by microencapsulation were studied to determine what
criteria must be satisfied to provide a protective immune response
to hepatitis B surface antigen (HBsAg) after a single injection of
vaccine. In mouse studies, the 50% effective dose (ED.sub.50) for
the alum precipitated Heptavax B vaccine (Merck, Sharp and Dohme)
was 3.8 ng when administered in a 3 injection regimen, but was 130
ng when one inmmunizing dose was used. Antigen release studies
revealed that HBsAg is bound tightly to the alum, indicating that
the antigen remains in situ until scavenged by phagocytic cells.
the ED.sub.50 with a 3 dose regimen of aqueous HBsAg was 180 ng, a
opposed to over 2000 ng for daily injections of low doses for 90
days and 240 ng for a regimen that employed initially high doses
that decreased geometrically at 3 day intervals over 90 days. The
ED.sub.50 was 220 ng for a single dose regimen of HBsAg
microencapsulated in poly (DL-lactide-co-glycolide) in a form that
was too large to be phagocytized and had an antigen release profile
similar to that achieved with the geometrically decreasing regimen
of doses. This indicates that single injection of microencapsulated
immunogens can achieve similar effects in vivo to those achieved
with multiple dose regimens. For HBsAg the effect to be achieved
appears to be 3 pulses of particulate immunogens that can be
scavenged by phagocytes.
Introduction
A major disadvantage of inactivated vaccines lies in their
inability to confer lasting immunity. Due to rapid elimination from
the body, multiple doses and boosters are usually required for
continued protection.sup.37. Alum adjuvants, achieving their
effects by mechanisms of antigen presentation and sustained antigen
release.sup.38, have been used successfully to increase the potency
of several inactivated vaccines including those against tetanus,
anthrax, and serum hepatitis.sup.39,40. Though useful, alum
preparations are deficient in several aspects. Control over
quantity and rate of antigen release is limited, often resulting in
a continued requirement for immunization schedules consisting of
multiple injections given over a period of several months to years.
Alum adjuvants are also non-biodegradable and thus remain within
the body, serving as a nidus for scar tissue formation.sup.38 long
after they have served their function.
Protracted, multiple immunization schedules are unacceptable during
massive mobilization and deployment of troops. Changing global
disease patterns and deployment of new biological warfare agents by
enemy forces require flexibility in the number and types of vaccine
antigen administered to soldiers departing for combat. Any
immunization schedule requiring completion during engagement in
non-linear combat would compromise this flexibility and place an
unreasonable burden on our health care delivery system.
The main objective of this study was, therefore, to develop a
biodegradable, controlled-release adjuvant system capable of
eliminating the need for multistep vaccination schedules. This
investigation was designed to: (1) determine in an animal model
hepatitis B vaccine release rate characteristics desirable for
single-step immunization, (2) incorporate those release rate
characteristics into a one-step biodegradable
poly(DL-lactide-co-glycolide)(DL-PLG) microencapsulated hepatitis B
surface antigen (HBsAg) vaccine, and (3) conduct an in vivo trial
comparing the effectiveness of this single-step vaccine against the
conventional three-step hepatitis vaccine currently
employed.sup.41. The results were intended to provide the
foundation for further development of single step vaccines against
hepatitis and other militarily significant diseases.sup.42.
Materials and Methods
Vaccine potency assay. Due to its availability, compatibility with
cage mates, and potential application to the study of hepatitis B
vaccine.sup.43, the female Walter Reed (ICR) stain mouse was used.
A hepatitis B vaccine potency assay for comparing the six-month
immunization schedule currently in use.sup.41 with that of a
single-step immunization by sustained antigen release was
established according to the following protocol: Specimens for
baseline antibody titers were collected from twenty mice by
exsanguination. Immediately prior to exsanguination, all mice
employed in this and other exsanguination procedures in these
studies were anesthetized with a 0.1 ml injection of V-Pento.
Groups of 12 mice were then immunized according to a schedule
consisting of either 0.25 ug, 0.025 ug, 2.5 ng, 0.25 ng, 2.5 pg, or
0.25 pg Heptavax B vaccine (HBV) administered in 50 microliter
volumes subcutaneously (s.c.) at the beginning and end of the
first, and end of the second month of the protocol. Antibody
responses to the vaccine were monitored immediately before the
third injection and approximately one month after the third
injection. Specimens for antibody determination were collected by
exsanguination of seven anesthetized mice from each group and
assayed along with the baseline samples by the Abbott Ausab
radioimmunoassay. Percent seroconversion verses micrograms vaccine
employed with calculated by the method of Reed and Muench.sup.43.
These data were employed to establish a mouse vaccine potency assay
calibrated to detect differences between Heptavax B and other forms
of hepatitis b vaccine.
In vitro antigen release rate from Heptavax B vaccine. Antigen
release from aluminum hydroxide adjuvant in HBV was measured by
pumping 2 cc per hour of 1:20,000 thimerosal in saline at 4.degree.
C. across a 0.2 u pore diameter Acrodisc filter apparatus
containing 20 ug of vaccine. The effluent, collected by a Gilford
fraction collector, was assayed periodically over several weeks for
protein by UV absorption at 280 nm on a Beckman model 25 double
beam spectrophotometer, and for HBsAg by the Abbot Ausria II
radioimmunoassay made quantitative by using HBsAg standards
supplied by Merk, Sharp, and Dohme. Accuracy of the HBsAg standards
were verified by Biuret protein determination and by UV absorbance
at 215 nm and 225 nm.sup.44. Nonspecific antigen retention on the
Acrodisc filter was assessed by measuring percent recovery of a
known quantity of HBsAg. Spontaneous degradation of vaccine antigen
was monitored by comparing daily rations of antigen to total
protein detected in the effluent.
Evaluation of HBsAg stability. These studies were designed to
characterize the stability of the aqueous antigen to the various
physical conditions employed in the microencapsulation process.
Conditions tested included lyophilization with reconstitution in
distilled water, cyclohexane, methylene chloride, chloroform,
methyl alcohol, acetone, iso-octane, hexane, acetone, pentane, or
heptane; irradiation while lyophilized; and, exposure to elevated
temperatures. Samples exposed to organic solvents were first
lyophilized, reconstituted with the test solvent, evaporated to
dryness under nitrogen at room temperature and reconstituted with
distilled water. Test samples were compared against untreated
controls by assaying serial dilutions of each with the Abbot Ausria
II procedure and comparing the plots of counts per minute verses
dilution.
Assessment of the effect of antigen release rate on vaccine
potency. Three regimens simulating patterns of free HBsAg release
that could be achieved by microencapsulation were contrasted with
the three monthly dose regimen of Heptavax B for immunizing mice.
To do so, 24 ICR mice were divided into groups and vaccinated as
indicated below. Seven mice from each subgroup were exsanguinated
at the end of the second and third months of the experiment. The
sera were separated and assayed for specific antibody response to
HBsAg by Abbot Ausab procedure.
HV regimen a: 14 mice/treatment receiving 3 s.c. injections of 250,
25, 2.5 or 0.25 ng doses of HBV a month apart.
HBsAg regimen a: 14 mice/treatment receiving 3 s.c. injections of
250, 25, 2.5 or 0.25 ng doses of aqueous HBsAg a month apart.
HBsAg regimen b: 14 mice/treatment receiving total doses of 750,
75, 7.5 or 0.75 ng of aqueous HBsAg over 3 months by s.c.
injections of ZX.sub.y ng at 3 day intervals, where Z is the total
dose, y is the injection number, and X is the fraction indicated on
the graph in FIG. 1 minus the fraction for the previous
injection.
HBsAg regimen c: 14 mice/treatment receiving daily s.c. injections
of 8.33, 0.833, 0.0833 or 0.00833 ng of aqueous HBsAg for 3
months.
Microencapsulation in DL:PLG. Microencapsulated immunogens were
fabricated by Southern Research Institute, Birmingham, Ala. DL-PLG
polymers were synthesized from the cyclic diesters, DL lactide and
glycolide, by using a ring-opening melt polymerization catalyzed by
tetraphenyl tin.sup.45. The resulting polymer was dissolved i
methylene chloride, filtered free of insoluble contaminants and
precipitated in methanol. Lactide-co-glycolide mole ration of the
product was determined by nuclear magnetic resonance spectroscopy.
Encapsulation of HBsAg in DL:PLG polymer was achieved by an organic
phase separation process.sup.46. Microcapsules of the desired size
(approximately 100 micron diameter in these studies) were isolated
from each batch by wet sieving with hexane through standard mesh
stainless steel sieves and then dried for 24 hours in a vacuum
chamber maintained at room temperature.
In vitro analysis of encapsulated antigens. Integrity of
encapsulated antigen was assessed by comparing the antigen to total
protein ratios present in microcapsule hydrolysates with those
obtained from suspensions of pure unencapsulated antigen.
Centrifuge tubes containing 1 ug of either microencapsulated or
pure vaccine antigen in 1 ml saline were incubated at 4.degree. C.
with shaking. Samples were collected at weekly intervals by
interrupting the incubation, sedimenting the contents of the tubes
by centrifugation and withdrawing the supernates. Sediments were
resuspended in 200 microliters of saline and supernates were
assayed for HBsAg by the Abbott Ausria II radioimmunoassay. The
HBsAg standard described earlier in this report was used as the
calibrator. Antigen destruction due to the encapsulation procedure
was monitored by a comparison between the antigen assayed from the
hydrolysate and from the untreated antigen control.
Assessment of the potency of DL:PLG microencapsulated HBsAg for
immunizing ICR mice when used alone and in combination with
Heptavax B vaccine. HBsAg loaded microcapsules that had been
fabricated by Southern Research Institute to release the majority
of their HBsAg load within 40 to 50 days were serially diluted in
10-fold steps by mixing the dry, loaded capsules with blank placebo
capsules of similar size and composition. The resulting stock and
diluted microcapsule preparations were placed onto lyophilizer when
not in use in order to assure minimum spontaneous degradation prior
to injection. On the day of injection, a predetermined weight of
microcapsules or placebo-diluted microcapsules was added to each
syringe. Immediately prior to injection either one or two ml of
injection vehicle (2 wt % carboxymethyl cellulose and 1 wt &
Tween 240 in water, Southern Research Institute) were drawn into
the microcapsule-loaded syringes, mixed and injected. All mice were
vaccinated s.c. as indicated below:
Group 1: 14 mice/treatment receiving 25, 25, 2.5, 0.25 or 0.925 ng
HBV.
Group 2: 14 mice/treatment receiving 1000, 250, 25 or 2.5 ng
aqueous HBsAg with Bovine Serum Albumin (BSA).
Group 3: 7 mice receiving 1600 ng microencapsulated HBsAg (HBsAg)
plus 0.25 ng HBV and 14 mice/treatment receiving 160, 16, 1.6 or
0.16 ng HBsAg plus 0.25 ng HBV.
Group 4: 7 mice receiving 1600 ng HBsAg plus 2.5 ng HBV and 14
mice/treatment receiving 160, 16, 1.6 or 0.16 ng HBsAg plus 2.5 ng
HBV.
Group 5.: 7 mice receiving 1600 ng HBsAg plus 25 ng HBV and 14
mice/treatment receiving 160, 16, 1.6 or 0.16 ng HBsAg plus 25 ng
NBV.
Group 6: 7 mice receiving 2500 ng HBsAg and 14 mice-treatment
receiving 250, 25, 2.5 or 0.25 ng HBsAg. Fifty-three days after
receiving the above injections, the mice were anesthetized with an
0.1 cc injection of V-Pento and exsanguinated. Blood samples were
allowed clot and the sera were separated by centrifugation. The
serum samples were assayed for antibody to HBsAg by the Abbott
Ausab procedure.
Results
Heptavax B vaccine potency. As can be seen from Table 4, the total
dose of vaccine which produced seroconversion in 50% of
TABLE 12 Potency of Heptavax B vaccine in ICR mice. No. ng Heptavax
B per Injection ED.sub.50 Inj. 250 25 2.5 .25 .025 .0025 .00025 ng
2 5/5 4/4 3/6 2/6 0/5 1/4 0/4 1.7 3 6/6 6/6 4/6 1/6 0/6 1/6 1/6 2.0
*Number positive seroconversions per number vaccinated. The
vaccinated mice (ED.sub.50) for HBV was approximately 2 ng, whether
the vaccine was given in 2 or 3 injections.
In vitro antigen release rate from HBV. HBsAg release from the 20
ug of Heptavax was not detected in any of the 21 fractions of
saline collected from the Acrodisc polycarbonate filter over a 30
day period. The lower limit of detection for the Abbott Auria II
assay employed was approximately 4.8 ng/ml. The Acrodisc filter
used in the antigen release study was back-washed with 10 mls
normal saline. Quantitation of the HBsAg present within this
back-wash eluent revealed the presence of the original 40 ug of
Heptavax vaccine which had been loaded into the filter at the start
of the experiment. This is the concentration which one would expect
to obtain if there had been no deterioration of the original 40
ug/ml HBsAg loaded onto the filter, none of the antigen eluted from
the alum adjuvant, and none of the vaccine had adsorbed onto or
passed through the filter.
Evaluation of antigen stability. Considerable effort was expended
in assessing the effects of physical conditions on the antigenicity
of HBsAg to insure that the conditions used for microencapsulation
would not cause serious degradation of the immunogen. Since
microencapsulation must be performed on dried materials which are
suspended in organic solvents, the HBsAg, which was provided as a
solution, had to be lyophilized. Initial attempts at lyophilizing
HBsAg in normal saline resulted in a total loss of detectable
antigen within samples. Dilution of the HBsAg sample 1:10 in
distilled water prior to freezing resulted in reservation of nearly
100% of the antigen detectable in the original sample. Studies of
antigen stability at elevated temperature revealed that HBsAg may
be heated to 50.degree. C. for up to one hour without appreciable
loss of antigen. The studies involving exposure of lyophilized
antigen to organic solvents indicated that iso-cane and hexane had
minimal effects on antigenicity, but that 95% to 100% of
antigenicity was lost upon exposure to either methylene chloride,
chloroform, cyclohexane, or methyl alcohol. Moderate antigen loss
occurred in the presence of acetone, pentane and heptane. As a
result of these studies, hexane was chosen as the solvent for
microencapsulation.
Assessment of the effect of antigen release rate on vaccine
potency. The results (Table 13) indicated that immunogen formation
(i.e., the alum adjuvant of Heptavax B) had far more
TABLE 13 Effect of immunogen formulation and vaccination regimen on
potency for immunizing ICR mice. Immunogen ng Total Dose HBsAg
ED.sub.50 Formulation Regiment 750 75 7.5 .75 ng Heptavax B a 7/7*
6/6 5/7 1/7 3.8 Aqu. HBsAg a 4/6 3/7 0/7 0/6 180 Aqu. HBsAg b 6/7
0/7 1/7 0/7 240 Aqu. HBsAg c 1/7 0/7 0/7 0/7 >2000 *Number
positive seroconversions per number vaccinated. a 3 injections of
1/3 total dose a month appart. b Injections administered every
three days for 90 days in decreasing dosages according to a
logarithmic progression. c Injections of 1/90 total dose daily for
90 days.
effect on potency than did the vaccination regimen, and that
pulsing with large doses of immunogen was more effective than
continuous administration of small doses.
HBsAg release from DL:PLG microcapsules. The microcapsules employed
in this study were designed to disintegrate within three weeks
after hydration. It is evident from the release curve (FIG. 10)
that they performed as designed, releasing approximately 17% of
their total load in an initial pulse and approximately 7% of the
remaining available HBsAg over the first three weeks.
Assessment of the potency of DL:PLG microencapsulated HBsAg for
immunizing ICR mice when used alone and in combination with
Heptavax B vaccine. The results (Table 14) indicate that the
microencapsulated HBsAg had approximately the same immunogenicity
as did the Heptavax B. Neither immunogens were sufficiently potent
to effect with a singly injection seroconversion rates similar to
those achieved after three injections of Heptavax B (Table 12).
Only the immunogen
TABLE 14 Potencies of Heptavax B and microencapsulated HBsAg by
single injections S.C. when administered alone and in combination
to immunize ICR mice. Var. Dose ng Const. ng Variable Dose Var.
Dose Tot. Dose Immunogen Dose mHBsAg 2500 250 25 2.5 .25 ED.sub.50
ng ED.sub.50 ng Heptavax B 0 13/14* 8/14 4/14 0/13 130 130 Heptavax
B 0.16 11/13 4/14 1/14 1.7 1.8 Heptavax B 1.6 10/13 1/14 0/13 100
100 Heptavax B 16 3/14 1/14 1/14 >470 >490 Heptavax B 160
3/12 2/11 1/12 >370 >530 Heptavax B 1600 7/7 7/7 7/7 <0.8
1600 Mic. HBsAg 0 3/6 6/15 1/13 2/10 2/14 220 220 *Number positive
seroconversions per number vaccinated.
combination of Heptavax B with 0.16 ng mHGsAg provided this level
of seroconversion. At the ED.sub.50 endpoint, the 0.16 ng dose of
mHGsAg is approximately 10% of the total dose. Similarly, a small
amount of Heptavax B appeared to enhance the immunogenicity of the
microencapsulated immunogen, although the combination was clearly
less immunogenic when the two formulations were present at
equivalent concentrations.
Discussion
The potential advantage of microcapsules lies in their ability to
be programmed during fabrication into forms that have quite
difference release profiles, including slow and steady release,
multiple bursts of antigen over a period of time, or combinations
of release forms. Sieving allows choice of microcapsule size, and
the ability of DL-PLG to sequester antigen from the host's immune
system until release occurs enhances control over exposure of the
recipient's immune system to antigen over a sustained period of
time. These characteristics provided the impetus for these studies
as they indicate potential for achieving the effects of a multiple
injection regimen by controlling release in vivo after a single
injection.
The results of these studies are important for gaining an under
standing of the fundamental differences between the manner in which
alum and microcapsules interact with the immune system. The antigen
release studies showed that alum firmly bound the antigen on its
surface, whereas the microcapsules sequestered the antigen load
within the interstices of an immunologically inert polymer. Release
of antigen from microcapsules was spontaneous and gradual while
antigen release from alum was probably enzymatically mediated
within host macrophages. Alum thus performed at least two useful
functions as an adjuvant: by bearing its entire load of antigen
upon its surface, it provided a large single exposure of antigen to
the host; and, by being readily phagocytized by host macrophages,
it served as a means of targeting the antigen to the immune
system.
In order for microcapsules to be efficacious as a vaccine delivery
system, a means of incorporating the two properties common to alum
adjuvant must be devised. These properties, which where discussed
above, are targeting antigen to the immune system and delivering
the antigen load in a single concentrated pulse at its target. A
gradual, sustained release of free antigen, as was achieved with
the 100 micron microcapsules used in these studies, could be
expected to elicit an immune response similar to that seen with
either regimen b or regimen c (Table 13), where multiple injections
of small doses were employed. In fact, as shown in Table 11, the
microencapsulated immunogen elicited a response similar to that
achieved with regimen b. This is probably due to the fact that the
microcapsules release approximately 10% of their antigenic load
immediately after injection.
Microcapsules with extended release patterns tend to be large
(>10 microns in diameter) and thus fail to be readily
phagocytized. In order for the larger microcapsules with prolonged
antigen release characteristics to be efficacious, the antigen
eventually released from those microcapsules would have be in a
form which targeted and concentrated it within the recipient's
immune system. This might be effectively achieved by
microencapsulation of antigen coated alum or by microencapsulating
clusters of smaller (<10 microns) microcapsules.
Microcapsules under 10 microns in diameter tend to be readily
phagocytized and also tend to under go rapid spontaneous
degradation due to their high surface to volume ratio. These
smaller microcapsules would be well suited for eliciting a primary
response if their pulse of antigen release could be programmed to
occur after phagocytosis.
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fifty percent endpoints. Amer. J. Hyg. 27:493-497.
44. Bradford, M. 1976. A rapid an sensitive method for the
quantitation of microgram quantities of protein utilizing the
pracile of protein-dye binding. Anal. Biochem. 72:248-254.
45. Jackanicy, T. M, et al. 1983. Polylactic acid as a
biodegradable carrier for contraceptive steroids. Contraception
8:227-234.
11. Kulkarni, R. K., E. G. Morre, A. F. Hegyeli, and F. Leonard.
1971. Biodegradable poly (lactic acid) polymers. J. Biomed. Mater.
Res. 5:169-181.
46. Cutright D. E., P.Bienvenido, J. Beasley, III, W. T. Larson,
and W. R. Posey. 1974. Degrdation rates of polymers and copolymers
and polyglycolic acids. Oral Surg. 37:142-152.
Phase III
This phase of the invention relates to providing novel
biocompatible and biodegradable microspheres for burst-free
programmable sustained release of biologically active agents,
inclusive of polypeptides, over a period of up to 100 days in an
aqueous physiological environment. Potentially release period is
capable of being further modulated beyond 100 days to about 365
days by careful selection of a blend of uncapped and end-capped
biodegradable-biocompatible copolymer and molecular weights.
Several publications and patents are available for sustained
release of active agents from biodegradable polymers, particularly,
poly(lactide/glycolides) (PLGA). Prior usages of PLGA for
controlled release of polypeptides have involved the use of molar
ratios of lactide/glycolide (L/G) of 75/25 to 100/0 for molecular
weights <20,000. Further prior art preparations of PLGA utilized
fillers or additives in the inner aqueous layer to improve the
stability and encapsulation efficency and/or to increase the
viscosity of the aqueous layer, thereby modulating polymer
hydrolysis and the biologically active agent or polypeptide
release.
In addition, the prior art use of PLGA copolymers were end-capped,
in that the terminal carboxyl end groups were blocked. In these
end-capped co-polymers, the microcapsule preparations exhibited a
low to moderate burst release of -10-40% of the entrapped
polypeptide in the first 24 hours after placement in an aqueous
physiological environment. In part, these characteristics are due
to the use of fillers in the inner aqueous phase. Further, a
1-month release of polypeptide is known with the use of a 75/25
co-polymer of PLGA of Mw <20,000.
Investigations in controlled release research has been proceeding
especially to obtain a 1 to 2 month delivery system for
biologically active agents or polypeptides using
poly(lactide/glycolide) polymers. However, most of these systems
have one or more of the following problems: Poor encapsulation
efficency and large `burst release` followed by an intermediate `no
release` or `lag phase` until the polymer degrades. In general,
release from these polymers occur over a period from about 4 weeks
to about several months. In addition, in order to achieve this
release a 50/50 copolymer of MW>30,000 or a 75/25 copolymer of
Mw>10,000 are employed which often results in residual polymer
remaining at the site of administration long after the release of
active core.
This invention provides biocompatible and biodegradable
microspheres that have been designed for novel, burst free,
programmable sustained release of biologically active agents,
including polypeptides over a period of up to 100 days in an
aqueous physiological environment.
Unlike currently available release systems, which rely on the use
of fillers/additives such as gelatin, albumin, dextran, pectin,
polyvinyl pyrrolidone, polyethylene glycol, sugars, etc., and are
still prone to low encapsulation efficiencies and "burst effects",
this invention achieves high encapsulation and "burst-free" release
without the use of any additive. In this invention, burst-free,
programmable sustained release is achieved through the use of a
unique blend of the `uncapped` and end-capped forms of
poly(lactide/glycolide) polymer in the molecular weight range of
2,000 to 60,000 daltons.
In general, microspheres described in this invention are produced
by a unique emulsification technique wherein an inner water-in-oil
(w/o) emulsion is stabilized by dispersing in a solvent-saturated
aqueous phase containing an emulsion stabilizer. A ternary w/o/w
emulsion is then formed by emulsifying the above w/o emulsions in
an external pre-cooled aqueous phase containing an o/w emulsifier.
Essentially, the inner w/o emulsion is comprised of an aqueous
layer containing from .about.2 to about 20% (w/w) of the active
agent to be entrapped and an oil layer containing
poly(lactide/glycolide) copolymer in concentrations ranging from
.about.5 to about--50% (w/w oil phase). The copolymer includes
molecular weight ranging from 2,000 to about 60,000 daltons, with
molar composition of lactide/glycolide from 90/10 to 40/60 and a
blend of its uncapped and end-capped forms in a ratio of 100/0 to
1/99. Very high encapsulation efficiencies of about 80 to 100% are
achieved depending on polymer molecular weight and structural
form.
Programmable release of active core over variable durations between
1-100 days is achieved by a judicious selection of process
parameters such as polymer concentration, peptide concentration and
the aqueous/oil phase ratio.
This invention is particularly suitable for high encapsulation
efficiencies and burst-free, continuous programmable release of
polypeptides of molecular weights ranging from 1,000 to about
250,000 daltons, and also other biologically active agents over a
period of 1-100 days. A uniqueness of the invention is that when
using a 100/0 blend of the uncapped and capped polymer, the final
phase of active core release is concurrent with the complete
solubilization of the polymer to innocuous components, such as
lactic and glycolic acids. This is a significant advantage over the
currently available 30 day--release systems wherein a major
regulatory concern is about toxicity of residual polymer at the
site of administration, long
This invention relates to the design of biocompatible and
biodegradable microspheres for novel, programmable sustained
release of biologically active agents, including polypeptides over
a period of up to 100 days in an aqueous Physiological environment
with little or no burst release.
Unlike currently available release systems which rely on the use of
fillers/additives such as gelatin, albumin, dextran, pectins
polyvinyl pyrrolidone, polyethylene glycol, sugars, etc., and are
still prone to low encapsulation efficiencies and "burst effects",
this invention achieves high encapsulation efficiency after release
of the active core.
The microcapsules described in this invention are suitable for
administration via several routes such as parenteral (intramuscular
subcutaneous), oral, topical, nasal, rectal and vaginal routes. and
`burst-tree` release without the use of any additive. In this
invention, burst-free, programmable sustained release is achieved
through the use of a unique blend of the `uncapped` and end-capped
forms of poly (lactide/glycolide) polymer.
The `uncapped` form refers to "poly(lactide/glycolide) with free
carboxyl end groups" which renders the polymer more hydrophilic
compared to the routinely used end-capped form. Currently used
`end-capped` polymer hydrates between 4-12 weeks depending on the
molecular weight, resulting in an intermediate `no release` or a
`lag phase`. The uncapped polymer hydrates typically between 5 to
60 days depending on the molecular weight, thus releasing its core
continuously without a lag phase. A careful blend of the two forms
and appropriate molecular weights and L/G ratios, results in a
continuous release between 1 to 100 days. In addition, release
within this time is programmable by a judicious selection of
process parameters such as polymer concentration, peptide
concentration and the aqueous/oil phase ratio.
The coploymer in this invention includes molecular weight ranging
from 2,000 to 60,000 daltons, a lactide/glycolide ratio of 90/10 to
40/60 and a blend of the uncapped/capped forms in the ratio of
100/0 to 1/99. The molecular weight of the polypeptide may be in
the range of 1000 to 250,000 daltons while that of other
biologically active agents may range from 100 to 100,000
daltons.
Microcapsules described in this invention are prepared by a unique
aqueous emulsification techinique which has been developed for use
with the uncapped polymer to provide superior sphere morphology,
sphere integrity and narrow size distribution. This is accomplished
by first preparing an inner water-in-oil (w/o) by mixing the
solutions of polymer in an organic solvent such as methylene
chloride and the biologically active agent in water. This is
followed by stabilization of the w/o emulsion in a
solvent-saturated aqueous solution containing an o/w emulsifier
such as polyvinyl alcohol. A ternary emulsion is then formed by
emulsifying the w/o emulsion in an external aqueous phase
containing the same emulsifier as above at concentrations ranging
from 0.25-1% w/v. Microcapsules are hardened upon solvent removal
by evaporation, rinsed to remove residual emulsifier and
lyophilized. Low temperature is used both at the time of primary
emulsification (w/o emulsion formation) and during the formation of
the final w/o/w emulsion to achieve stable emulsion and superior
sphere characteristics.
In the context of the invention, a biologically active agent is any
water-soluble hormone drugs, antibiotics, antitumor agents,
antiinflammatory agents, antipyretics, analgesics, antitussives,
expectorants, sedatives, muscle relaxants, antiepileptics,
antiulcer agents, antidepressants, antiallergic drugs,
cardiotonics, antiarrhythmic drugs, vasodilators,
antihypertensives, diuretics, anticoagulants, antinarcotics, and
the agents listed in the summary of the invention section
herein
More precisely, applicants have discovered a pharmaceutical
composition and process with the following itemized features:
1. A controlled release microcapsule pharmaceutical formulation,
which may contain a pharmaceutically-acceptable adjuvant, for
burst-free, sustained, programmable release of a biologically
active agent over a duration from 1-100 days, comprising an active
agent and a blend of uncapped and end-capped biodegradable
poly(lactide/glycolide).
2. The pharmaceutical formulation of item 1, wherein the
biodegradable poly(lactide/glycolide) is a blend of uncapped and
capped forms, in ratios ranging from 100/0 to 1/99.
3. The microcapsules of items 1 or 2 wherein the copolymer (lactide
to glycolide L/G) ratio for uncapped and endcapped polymer is 52/48
to 48/52.
4. The microcapsules of items 1 or 2 wherein the copolymer L/G
ratio for uncapped and end-capped polymer is 90/10 to 40/60.
5. The microcapsules of items 1 or 2 or 3 or 4 wherein the
molecular weight of the copolymer is between 2,000-60,000
daltons.
6. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein the
biologically active agent is a peptide or polypeptide.
7. The microcapsules of item 6, wherein said polypeptide is
histatin consisting of 12 amino acids and having a molecular weight
of 1563.
8. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6
characterized by the capacity to completely release histatin in an
aqueous physiological environment from 1-35 days with a 100/0 blend
of uncapped and end-capped poly(lactide/glycolide) having a L/G
ratio of 48/52 to 52/48, and a molecular weight <15,000.
9. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6
characterized by the capacity to completely release histatin in an
aqueous physiological environment from 18-40 days with a 100/0
blend of uncapped and end-capped poly(lactide/glycolide) having a
L/G ratio of 48/52 to 52/48 and a molecular weight range of
28,000-40,000.
10. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6
characterized by the capacity to release up to 90% of the histatin
in an aqueous physiological environment from 28-70 days with a
0/100 blend of uncapped and end-capped poly(lactide/glycolide)
having a L/G ratio of 48/52 to 52/48 and a molecular weight range
of 10,000-40,000 daltons.
11. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6
characterized by the capacity to release up to 80% of histatin in
an aqueous physiological environment from 56-100 days with a 0/100
blend of uncapped and end-capped poly(lactide/glycolide) having a
L/G ratio of 75/25 and a molecular weight of <15,000
daltons.
12. The microcapsules of items 7 or 8 or 9 or 10 or 11 having
analogs of histatin with chain lengths of from 11-24 amino acids of
molecular weights from 1,500-3,000 daltons and characterized by the
following structures:
1. D S H A K R H H G Y K R K F H E K H H S H R G Y
2. K R H H G Y K R K F H E K H H S H R G Y R
3. K R H H G Y K R K F H E K H H S H R
4. R K F H E K H H S H R G Y R
5. A K R H H G Y K R K F H
6. *A K R H H G Y K R K F H
7. K R H H G Y K R K F
* D-amino acid
13. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein the
biologically active agent is a polypeptide Leutinizing hormone
releasing hormone (LHRH) that is a decapeptide of molecular weight
1182 in its acetate form, and having the structure:
p- E H W S Y G L R P G
14. The microcapsule of items 6 or 7 or 8 or 9 or 10 or 11 or 12 or
13 having a molecular weight of from 1,000 to 250,000 daltons.
15. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12
or 13 or 14 wherein release profiles of variable rates and
durations are achieved by blending uncapped and capped microspheres
as a cocktail in variable amounts.
16. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12
or 13 or 14 wherein release of profiles of variable rates and
duration are achieved by blending uncapped and capped polymer in
different ratios within the same microshreres.
17. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12
or 13 or 14 or 15 or 16 wherein the entrapped polypeptide is any of
the vaccine agents against enterotoxigenic E. coli (ETEC) such as
CFA/I,CFA/II,CS1,CS3,CS6 and CS17 and other ETEC-related
enterotoxins.
18. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11 or 12
or 13 or 14 or 15 or 16 or 17 wherein the entrapped polypeptide
consists of peptide antigens of molecular weight range of about
800-5000 daltons for immunization against enterotoxigenic E. coli
(ETEC).
19. The microcapsules of items 1 or 2 or 3 or 4 or 5 wherein said
biologically active agents are selected from the group consisting
of water-soluble hormone drugs, antibiotics, antitumor agents, anti
inflammatory agents, antipyretics, analgesics, antitussives,
expectorants, sedatives, muscle relaxants, antiepileptics,
antiulcer agents, antidepressants, antiallergic drugs,
cardiotonics, antiarrhythmic drugs, vasodilators,
antihypertensives, diuretics, anticoagulants, and antinarcotics, in
the molecular weight range of 100-100,000 daltons.
20. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8
wherein said biodegradable poly(lactide/glycolide) is in an oil
phase, and is present in about 1-50% (w/w).
21. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9
or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 wherein
concentration of the active agent is in the range of 0.1 to about
60% (w/w).
22. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9
or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 wherein a ratio of
the inner aqueous to oil phases is about 1/4 to 1/40(v/v).
23. A process for preparing controlled release microcapsule
formulations characterized by burst-free, sustained, programmable
release of biologically active agents comprising: Dissolving
biodegradable poly (lactide/glycolide), in uncapped form in
methylene chloride, and dissolving a biologically active agent or
active core in water; adding the aqueous layer to the polymer
solution and emulsifying to provide an inner water-in-oil (w/o)
emulsion; stabilizing the w/o emulsion in a solvent-saturated
aqueous phase containing a oil-in-water (o/w) emulsifier; adding
said w/o emulsion to an external aqueous layer containing
oil-in-water emulsifier to form a ternary emulsion; and stirring
the resulting water-in-oil-in-water (w/o/w) emulsion for sufficient
time to remove said solvent, and rinsing hardened microcapsules
with water and lyophilizing said hardened microcapsules.
24. A process for preparing controlled release microcapsule
formulations characterized by burst-free, sustained, programmable
release of biologically active agents comprising:
dissolving biodegradable poly(lactide/glycolide) in end-capped form
in methylene chloride, and dissolving a biologically active agent
or active core in water; adding the aqueous layer to the polymer
solution and emulsifying to provide an inner water-in-oil emulsion;
stabilizing the w/o emulsion in a solvent-saturated aqueous phase
containing a oil-in-water (o/w) emulsifier; adding said w/o
emulsion to an external aqueous layer containing oil-in-water
emulsifier to form a ternary emulsion; and stirring a resulting
water-in-oil-water (w/o/w) emulsion for sufficient time to remove
said solvent; and rinsing hardened microcapsules with water; and
lyophilizing said hardened microcapsules.
25. The process of items 23 or 24 wherein a solvent-saturated
external aqueous phase is added to emulsify the inner w/o emulsion
prior to addition of the external aqueous layer, to provide
microcapsules of narrow size distribution range between 0.05-500
.mu.m.
26. The process of items 23 or 24, wherein a low temperature of
about 0-4.degree. C. is provided during preparation of the inner
w/o emulsion, and a low temperature of about 4-20.degree. C. is
provided during preparation of the w/o/w emulsion to provide a
stable emulsion and high encapsulation efficiency.
27. The process of items wherein a 100/0 blend of uncapped and
end-capped polymer is used to provide release of the active core in
a continous and sustained manner without a lag phase.
28. The microcapsules of items 6, wherein, when the entrapped
polypeptide is active at a low pH, such as LHRH,
adrenocorticotropic hormone, epidermal growth factor, calcitonin
released polypeptide is bioactive.
29. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11,
wherein, when entrapped peptide such as histatin is inactive at a
low pH, a pH-stabilizing agent of inorganic salts are added to the
inner aqueous phase to maintain biological activity of the released
peptide.
30. The microcapsules of items 6 or 7 or 8 or 9 or 10 or 11
wherein, when entrapped polypeptide such as histatin is inactive at
a low pH, a non-ionic surfactant such as polyoxyethylene sorbitan
fatty acid esters (Tween 80, Tween 60 and Tween 20) and
polyoxyethylene--polyoxypropylene block copolymers (Pluronics) is
added to the inner aqueous phase to maintain biological activity of
the released polypeptide.
31. The microcapsules of items 29, wherein placebo spheres loaded
with the pH-stabilizing agents are coadministered with
polypeptide-loaded spheres to maintain the solution pH around the
microcapsules and preserve the biological activity of the released
peptide in instances where the addition of pH-stabilizing agents in
the inner aqueous phase is undesirable for the successful
encapsulation of the acid pH sensitive polypeptide.
32. The microcapsules of item 30 wherein placebo spheres loaded
with non-ionic surfactant are coadministered with
polypeptide-loaded spheres to maintain biological activity of the
released peptide where the addition of non-ionic surfactants in the
inner aqueous phase is undesirable for successful encapsulation of
the acid pH sensitive polypeptide.
33. The microcapsules of items 1 or 2 or 3 or 4 or 5 or 6 or 8 or 9
or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 comprising a blend
of uncapped and capped polymer, wherein complete solubilization of
the copolymer leaves no residual polymer at the site of
administration and occurs concurrently with the complete release of
the entrapped agent.
34. A process of using microcapsules of items 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
or 18 or 19 or 20 for human administration via parenteral routes,
such as intramuscular and subcutaneous.
35. A process of using microcapsules of items 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
or 18 or 19 or 20 for human administration via topical route.
36. A process of using microcapsules of items 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
or 18 or 19 or 20 for human administration via oral routes.
37. A process of using microcapsules of items 1 or 2 or 3 or 4 or 5
or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17
or 18 or 19 or 20 for human admininstration via nasal, transdermal,
rectal, and vaginal routes.
Conservation of Bioactivity of Polypeptides
As the polymer degrades rapidly, there is a precipitous drop in pH
accompanied by the release of soluble oligomers in the
microenvironment which may affect the biological activity of acid
pH-sensitive peptides/proteins. In such instances, biological
activity can be maintained by the use of inorganic salts or
buffering agents in the inner aqueous phase codissolved with the
peptide.
The following unique advantages are characteristics of this
invention:
1. Burst-free, prolonged, sustained release of polypeptides and
other biologically-active agents from biocompatible and
biodegradable microcapsules up to 100 days in an aqueous
physiological environment without the use of additives in the
core.
2. Release of active core programmable for variable durations over
1-100 days, by using a blend of uncapped and capped polymer of
different molecular weights and copolymer ratio, and by
manipulating the process parameters.
3. Complete release of the active core is concurrent with complete
solubilization of the carrier polymer to innocuous components, such
as lactic and glycolic acids, especially when using a 100/0 blend
of uncapped/capped polymer. This is of tremendous significance, as
most biodegradable polymers currently used for 1-30 day delivery,
do not degrade completely at the end of the intended release
duration, thereby causing serious concern of regulatory authorities
on the effects of residual polymer at the site of
administration.
4. Ease of administration of the microcapsules in various dosage
forms via several routes, such as parenteral (intramuscular and
sucutaneous), oral, topical, nasal, vaginal, etc.
The hydrophilic homo-and co-polymers based on D,L-lactide and
glycolide contains hydrophilic adjusted homo-and co-polymers with
free carboxylic end groups, and is characterized by the formula:
##STR1##
Wherein Z=Molecular Weight/130; for example Z=92 for Mw 12,000 and
262 for Mw 34,000.
While the molar ratio of the lactide to glycolide may vary, it is
most preferred that the lactide to glycolide copolymer ratio be
50:50.
Reference is now made to FIG. 48 which depicts a blood-drug
concentration versus time graph that shows conventional drug
administration using a series of dosages compared to an ideal
controlled release system. Unfortunately, many drugs have a
blending of the two forms in a single formulation comprising
different ratios of uncapped to capped polymer, would significantly
influence the polymer hydration and hence release of the active
core thereby providing release curves of any desirable pattern.
Manipulation of polymer hydration and degradation resulting in
modulation of release of active core is achieved by the addition of
uncapped polymer to end-capped polymer in amounts as low as 1% up
to 100%.
While referring to Table 14 in conjunction with FIG. 50, it can be
seen that the cumulative Histatin release from PLGA microspheres
from several batches prepared using 50/50 and 75/25 uncapped and
end-capped, polymer modulates release between 1 to 100 days by
varying the process parameters. 1-35 days by uncapped 50/50, 18-56
days by capped 50/50 and 56-100 days by capped 75/25.
In referring to FIG. 51, a view is provided through a scanning
electron micrograph of PLGA microspheres designed for a one to two
month release system prepared using end-capped polymer of Mw 30-40
k daltons.
FIG. 52 depicts the cumulative Histatin release from PLGA
microspheres, in which the release profiles are from several
batches prepared using 50/50, uncapped and capped polymer, and
varying the process parameters to modulate release between 28 to 60
days.
FIG. 53 represents cumulative Histatin release from PLGA
microspheres--these combined release profiles are from several
batches prepared using 50/50 uncapped and capped polymer, and
varying the process parameters to modulate release between 1-60
days.
In the context of the invention, a biologically active agent is any
water-soluble antibiotics, antitumor agents, antipyretics
analgesics, anti-inflammatory agents, antitussives, expectorants,
sedatives, muscle relaxants, anti epileptics, antiulcer agents,
anti-depressants, anti-allergic drugs, cardiotonics,
antiarrhythmics drugs, vasodilators, antihypertensives, diuretics,
anticoagulants, hormone drugs, anti-narcotics, etc.
In general, "burst free" sustained release delivery of biologically
active agents from PLGA microspheres is accomplished in the context
of this invention using of 90/10 to 40/60 molar ratios, and ratios
of uncapped polymer to end-capped polymer of 100/0 to 1/99.
In general, the approaches for designing the biologically active
agents encapsulated in the uncapped and combination
uncapped/end-capped PLGA microspheres and characteristics of these
encapsulants are briefly set forth below as follows:
1. Providing PLGA microspheres of surface morphologies using 50/50
uncapped and capped polymers of Mw .about.8-40K daltons as shown in
FIGS. 49 and 51.
2. Providing in vitro release of a polypeptide, Histatin from PLGA
microspheres, as shown in FIGS. 50 and 52, using uncapped and
capped polymer of Mw .about.8-40K daltons and molar ratios such as
50/50 and 75/25.
For example, design of a 1-12 week bioactive compound release
system is achieved using PLGA with the following
specifications:
1. Polymer molecular weight:--about 2-60K daltons
2. Copolymer molar ratio (L/G):--90/10 to 40/60
3. Polymer end groups:--uncapped and/or end-capped and combining
judiciously within the following parameters:
4. Polymer concentration--from 5 to 50%
5. Inner aqueous to oil phase ratio:--1:5 to 1:20 (v/v)
6. Peptide loads:--from 2 to about 40% (w/w polymer) and by using
the unique aqueous emulsification method described in the
invention.
The uniqueness and novelty of invention may generally be summarized
in a brief way as follows:
1. Use of uncapped poly(lactide/glycolide) to achieve burst-free,
continuous, sustained, programmable release of biologically active
agents over 1-100 days.
2. Use of a unique aqueous emulsification system to achieve
superior microsphere characteristics such as uniform sphere
morphology and narrow size distribution.
3. Burst-free, prolonged, sustained release of polypeptides and
other biologically actice agents from biocompatible and
biodegradable microcapsules up to 100 days in an aqueous
physiological environment without the use of additives in the inner
core.
4. Release of active core programmable for variable durations over
1-100 days by using a blend of uncapped and capped polymer for
different molecular weights and copolymer rations and manipulating
the process parameters.
5. Complete release of the active core concurrent with complete
solubilization of carrier polymer to innocuous components such as
lactic and glycolic acids, especially when using a 100/0 blend of
uncapped/capped polymer. This is of tremendous significance as most
biodegradable polymers currently in use for 1-30 day delivery, do
not degrade completely at the end of the intended release duration
causing serious concern for regulatory authorities on the effects
of residual polymer at the site of administration.
6. Ease of administration of the microcapsules in various dosages
forms via several routes such as parenteral (intramusclar and
subcutaneous), oral, topical, nasal, vaginal, etc.
The following examples are illustrative of, but not limitations
upon the microcapsule compositions pertaining to this
invention.
Example 12
Polylactide/glycolide (PLGA) microcapsules are prepared by a unique
aqueous emulsification technique which has been developed for use
with the uncapped polymer to provide superior sphere morphology,
sphere integrity and narrow size distribution (See FIG. 32 and
32a). This is accomplished by dissolving the polymer in a
chlorinated hydrocarbon solvent such as methylene chloride and
dissolving the biologically active agent in water. A w/o emulsion
is then formed by mixing the solutions of polymer and the active
agent by sonication, followed by emulsion stabilization in a
solvent--saturated aqueous solution containing polyvinyl alcohol. A
ternary emulsion is then formed by emulsifying the w/o emulsion in
an external, pre-cooled aqueous phase containing polyvinyl alcohol
(0.25-1% w/v). Microcapsules are hardened upon removal of solvent
by evaporation, rinsed to remove any residual emulsifier, and then
lyophilized.
Table 16 lists the microcapsule compositions, Nos. 1-21 thus
prepared, consisting of a biologically active polypeptide, Histatin
(composed of 12 amino acids and a molecular weight of 1563) and
blends of uncapped and capped polymer of ratios 100/0 to 1/99, and
having a lactide/glycolide ratio of 90/10 to 40/60, and a molecular
weight range between 2000 to 60,000 daltons.
Example 13
Microcapsule compositions are prepared as described in Example 1
wherein the copolymer L/G ratio is 48/52 to 52/48, and the ratio of
uncapped/capped polymer is 100/0. The active core is Histatin (Mw
1563), the polymer molecular weight is <15,000 and the polymer
concentrations vary from 7% to .about.40% w/w. Compositions 1,2,4
12-14 and 16-18 are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment, such as phosphate-buffered
saline, pH 7.0 maintained at 37.+-.1.degree. C. are plotted as
cumulative percentage release versus time, and presented in FIG.
50.
Burst-free, variable release from 1-35 days is achieved by varying
the polymer concentration from 7 to .about.40% w/w in the oil
phase.
Example 14
Microcapsule compositions are prepared as described in Example 2,
wherein the aqueous/oil ratio is varied from 1/4 to 1/20 (v/v).
Compositions 1,2,4 and 12 are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment described in Example are plotted
as cumulative percentage release versus time, and presented in FIG.
50.
Burst-free, continuous release from 1-35 days, with different onset
and completion times are achieved by selecting different w/o ratios
in the inner core.
Example 15
Microcapsule compositions are prepared as described in Example 2,
wherein the polymer molecular weight is 28,000-40,000 and polymer
concentrations vary from 5% to .about.15% w/w. Compositions 19-21
are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment are described in Example 2 are
plotted as cumulative percentage release versus time and presented
in FIG 52.
Burst-free, variable release from 18-40 days is achieved by varying
the polymer concentration.
Example 16
Microcapsule compositions are prepared as described in Example 2,
wherein the ratio of uncapped/capped polymer is 1/99 and polymer
concentrations vary between 5% to .about.12% w/w. Compositions 10
and 11 are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment are described in Example 2, and
plotted as cumulative percentage release versus time and presented
in FIG. 50.
Burst-free, variable release from 28-70 days is achieved by varying
the polymer concentration in the oil phase.
Example 17
Microcapsule compositions are prepared as described in Example 5,
wherein polymer molecular weight is 28,000-40,000 and polymer
concentrations vary between 5% to .about.12% w/w. Compositions 5
and 6 are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment are described in Example 2 and
are plotted as cumulative percentage release versus time, and
presented in FIG 52.
Burst-free, variable release from 28-70 days is achieved by varying
the polymer concentration.
Example 18
Microcapsule compositions are prepared as described in Example 6,
wherein the aqueous/oil ratio varies between 1/5 to 1/25 (v/v).
Compositions 3 and 7 are listed in Table 16.
Release profiles of the active core from the compositions in an
aqueous physiological environment are described in Example 2, and
plotted as cumulative percentage release versus time, and presented
in FIG. 52
Burst-free, variable release from 28-70 days is achieved by varying
the aqueous/oil ratios.
Example 19
Microcapsule compositions are prepared as described in Example 5,
wherein the copolymer ratio is 75/25 and polymer concentrations
vary between 5% to .about.25% w/w. Compositions 8 and 9 are listed
in Table 1.
Release profiles of the active core from the compositions in an
aqueous physiological environment are described in Example 2, and
are plotted as cumulative percentage release versus time, and
presented in FIG. 50.
Burst-free, variable release from 56->90 days is achieved by
varying the polymer concentration in the oil phase.
Example 20
Microcapsule compositions are described in Example 2, wherein the
active core is leutinizing hormone releasing hormone (LHRH, a
decapeptide of molecular weight 1182) and the polymer concentration
is .about.40% w/w. Release profiles of the active core from the
composition in an aqueous physiological environment is described in
Example 2, and is plotted as cumulative percentage release versus
time, and presented in FIG. 54.
Burst-free, continuous and complete release is achieved within 35
days, similar to Histatin acetate.
Example 21
Microcapsule compositions are prepared as described in Example 2,
wherein an additive such as sodium salt (carbonate or bicarbonate)
is added to the inner aqueous phase at concentrations of 1-10% w/w
to maintain the biological activity of the released
polypeptide.
Burst-free, variable release from 1-28 days is achieved similar to
Examples 2 & 3, and the released polypeptide is biologically
active until 30 days, due to the presence of the sodium salt.
Example 22
Microcapsule compositions are prepared as described in Example 2,
wherein an additive such as a nonionic surfactant,
polyoxyethylene/polyoxypropylene block copolymer (Pluronics F68 and
F127) is added to either the inner oil or the aqueous phase at
concentrations from 10-100% w/w, to maintain the biological
activity of the released polypeptide.
Burst-free, continuous release from 1-35 days is achieved similar
to Examples 2 & 3, and the released polypeptide is bioactive
due to the presence of the surfactant.
Example 23
Cumulative histatin release from the nicrocapsule Compositions
described in Examples 1 through 11 and release profiles plotted in
FIGS. 49 and 50 show the burst-free, programmable peptide release
for variable duration from 1-100 days. Virtually any pattern of
cumulative release is achievable over a 100 day duration by a
judicious blending of several compositions, as shown in FIG.
53.
TABLE 1 Ampicillin Anhydrate Microcapsules Evaluated in Rats
Antibiotic Microcapsule Dose/ In Vivo Microcapsule Core Loading,
Wound, g (Antibiotic Experiment Batch Wt Percent Equivalent, mg)
Efficacy A382-140-1 18.5 0.50 (92.50) Dose-Response A681-31-1 18.1
0.50 (90.50) I 0.25 (45.25) 0.10 (18.10) 0.05 (9.05) Dose-Response
B213-66-1S 11.4 0.25 (28.50) II 0.15 (17.10) 0.05 (5.70)
TABLE 2 Effect of Immediate Antibiotic Therapy for Prevention of
Experimental Osteomyelitis in a Rabbit Tibia Model Group Bacterial
Radiographic Positive Counts.sup.b Treatment Severity.sup.a Bone
Cultures A Parenteral therapy 0 0/6 0 for 14 days B
Microencapsulated 0.43 .+-. 1.13 0/7 0 ampicillin.sup.c C
Unencapsulated 0 1/4 1.2 (.+-.2.3) .times. 10.sup.2
ampicillin.sup.c D Placebo 7.00 .+-. 0.0 4/4 4.9 (.+-.8.3) .times.
10.sup.6 microcapsules.sup.c E Injection 6.67 .+-. 0.58 4/4 1.3
(.+-.0.7) .times. 10.sup.6 vehicle.sup.c F No treatment 5.25 .+-.
2.06 5/5 2.0 (.+-.4.5) .times. 10.sup.7 .sup.a Mean radiographic
severity score at 7-weeks post treatment. .sup.b Mean (.+-.
standard deviation) CFU of S. aureus recovered per gram of bone.
.sup.c Intramedullary injection.
TABLE 3 Effect of Delayed Therapy without Debridement for Treatment
of Experimental Osteomyelitis in a Rabbit Tibia Model Positive
Bacterial Goup Treatment Bone Cultures Counts.sup.b A Parenteral
therapy 6/8 5.9 (.+-.16.7) .times. 10.sup.6 for 14 days B
Microencapsulated 4/8 1.2 (.+-.2.2) .times. 10.sup.3
ampicillin.sup.c C Unencapsulated 5/7 2.6 (.+-.7.0) .times.
10.sup.5 ampicillin.sup.c D No treatment 6/6 2.8 (.+-.2.9) .times.
10.sup.5 .sup.a No statistically significant differences between
groups by Chi square analysis (p = 0.23) .sup.b Mean (.+-. standard
deviation) CFU of S. aureus recovered per gram of bone. .sup.c
Intramedullary injection.
TABLE 4 Effect of Delayed Therapy with Debridement for Treatment of
Experimental Osteomyelitis in a Rabbit Tibia Model Positive
Bacterial Goup Treatment.sup.a Bone Cultures Counts.sup.b A
Microencapsulated .sup. 0/10.sup.c 0 ampicillin B Unencapsulated
7/10 3.3 (.+-.4.8) .times. 10.sup.2 ampicillin.sup.c C Placebo 5/5
9.1 (.+-.10.9) .times. 10.sup.4 microcapsules D Injection vehicle
5/5 3.7 (.+-.4.9) .times. 10.sup.5 .sup.a All substances were
implanted locally into the medullary canal at the time of
debridement. .sup.b Mean (.+-. standard deviation) CFU of S. aureus
recovered per gram of bone. .sup.c significantly different (p <
0.01) from all other groups by Chi square analysis.
TABLE 5 Survival of E. coli and S. aureus in rat soft-tissue at 28
days following local or systemic treatment with cefazolin. Mean
(.+-.sd) Log CFU/g tissue Contamination Treatment Group (N) Dose E.
coli S. aureus Rate A: CZ microspheres (6) 50 mg 1.01 .+-. 1.59
0.50 .+-. 1.21 2/6 (33%) B: CZ microspheres (6) 250 mg 0.91 .+-.
1.41 0.42 .+-. 1.04 2/6 (33%) C: CZ microspheres (6) 500 mg 0 0 0/6
(0%) D: Free CZ powder (6) 110 mg 0.57 .+-. 1.40 0.53 .+-. 1.29 1/6
(17%) E: Systemic CZ (6) 30 mg/kg 4.44 .+-. 0.91 0.83 .+-. 2.03 6/6
(100%) F: No treatment (3) 0 4.28 .+-. 0.34 2.12 .+-. 1.83 3/3
(100%)
TABLE 6 Effect of early antibiotic therapy on infection in S.
aureus contaminated rabbit tibial fractures stabilized with
internal fixation. No. of Animals with: Mean (.+-.SD) Deep Positive
Bone log bacteria Treatment Group (N) Infection Cultures (CFU/g) A:
CZ microspheres (7) 0/7 1/7 0.3 .+-. 0.9 B. CZ powder (6) 0/6 1/6
0.2 .+-. 0.5 C. Systemic CZ (5) 3/5 4/5 3.0 .+-. 2.1 D. Placebo
microspheres (3) 3/3 3/3 5.2 .+-. 0.2 E. No treatment (4) 3/4 4/4
4.2 .+-. 0.5 Rabbit fracture-fixation model. Table 6 shows the
results of the clinical and bacteriological findings at 8 weeks in
25 surviving rabbits when local or systemic antibiotic therapy with
cefazolin was initiated within 30 minutes following bacterial
contamination of the fractures. Deep infection, defined as the
presence of pus on the fixation plate or in the deep tissues, # was
noted in 6 of the 7 (86%) control animals in Group D (placebo
microspheres) and Group E (no treatment). Cultures of the tibiae
from all 7 controls were positive for S. aureus. Of the 5 surviving
Group C animals who received a 1 week course of systemic cefazolin
therapy, deep infection was noted in 3 cases and S. aureus was
recovered from the bones of 4 of the 5 animals. # In contrast, no
clinical evidence of infection was detected in any of the 7 Group A
animals who received local antibiotic therapy with CZ microspheres
or in the 6 animals in Group B who received an equivalent local
dose of free CZ powder. Cultures of the tibiae were sterile in 6 of
7 (86%) Group A and 5 of 6 (83%) Group B animals, respectively.
There was a statistically significant # difference in the mean log
S. aureus counts of the Group A and Group B animals and all other
groups by analysis of variance (p < 0.05). The mean log S.
aureus counts for Group C was also significantly different from all
groups with the exception of Group E (no treatment).
TABLE 7 Effect of delayed antibiotic therapy on infection rates in
S. aureus contaminated rabbit tibial fractures No. of Animals with:
Mean (.+-.SD) Deep Positive Bone log bacteria Treatment Group (N)
Infection Cultures (CFU/g) A: CZ microspheres (8) 0/8 0/8 0 B. CZ
powder (8) 4/8 6/8 2.4 .+-. 1.8 E. No treatment (7) 5/7 7/7 4.3
.+-. 1.0 Table 7 shows the results of the clinical and
bacteriological findings at 8 weeks in 23 surviving rabbits when
local antibiotic therapy was delayed for 2 hours following
contamination of the fractures. Clinical evidence of infection was
present 5 of 7 (71%) control animals in Group C and cultures of the
tibiae yielded S. aureus in all 7 cases. Of the 8 animals in Group
B who received local antibiotic therapy with CZ powder, deep
infection was noted in 4 animals and S. aureus was recovered in 6 #
of 8 (75%) cases. In contrast, none of the 8 animals in Gorup A (CZ
microsperes) developed clinical infections and cultures of the
tibiae were sterile in all cases. One way analysis of variance
showed a statistically significant difference in the mean log S.
aureus counts between Groups A and B (p = 0.0014); Groups A and C
(p < 0.0001); and Groups B and C (p = 0.0269).
TABLE 8 Efficacy of Cefezolin Microspheres in Rat Soft Tissue
Wounds Contaminated with a Cefazoll-Resistant Strain of S. aureus
(MIC = 64 .mu.g/ml) Number of Number (%) Treatment Group Dose
Animals Sterile Wounds CZ microspheres .sup. 550 mg.sup.a 6 5/6
(83%) Free CZ powder 110 mg 6 6/6 (100%) Systemic CZ 30 mg/kg
.times. 7 days 6 0/6 (0%) Controls No antibiotics .sup. 3.sup.b 2/2
(0%) .sup.a 500 mg of CZ microspheres was applied to the wounds
representing 110 mg of cefazolin equivalent .sup.b One control
animal died during the experiment and no cultures were performed.
LEGEND: CZ microspheres = Cefazolin-loaded lactide-co-glycolide
microspheres Free CZ powder = Unencapsulated cefazolin powder
Systemic CZ = Intramuscular administration of cefazolin (30
mg/kg/day) given at 8 hour intervals for 7 consecutive days.
Controls = No antibiotic treatment.
TABLE 16 Microcapsule compositions containing Histatin polypeptide
Polymer Description L/G Mol. Wt. Conc in Theoretic peptide Internal
Phase Emulsification Composition # Ratio & Type (Mw .times.
10.sup.3) DCM (w/w) Core Load (%) Ratio (w/o) Technique 1. 50/50, U
12 38 5 1:20 A 2. 50/50, U 12 18.5 2 1:20 A 3. 50/50 34 10 5 1:20 A
4. 50/50, U 12 38 5 1:4 A 5. 50/50 34 7 5 1:10 B 6. 50/50 34 10 5
1:10 B 7. 50/50 34 10 5 1:10 A 8. 75/25 12 10 5 1:10 B 9. 75/25 12
23.5 5 1:10 B 10. 50/50 12 10 5 1:10 B 11. 50/50 12 7 5 1:10 B 12.
50/50, U 12 10 5 1:10 B 13. 50/50, U 12 7 2.3 1:10 B 14. 50/50, U
12 10 5 1:10 B 15. 50/50, U 34 10 5 1:10 B 16. 50/50, U 12 10 5
1:10 B 17. 50/50, U 12 20 5 1:10 B 18. 50/50, U 12 40 5 1:10 B 19.
50/50, U 34 5 5 1:10 B 20. 50/50, U 34 10 5 1:10 B 21. 50/50, U 34
15 5 1:10 B Acronyms: L/G ratio: Copolymer composition of
lactide/glycolide DCM: Methylene Chloride Mw: Molecular weight in
daltons A: w/o/w emulsification without an intermediate step for
emulsion stabilization B: w/o/w emulsification with an intermediate
step for emulsion stabilization U: Uncapped polymer
* * * * *